Method of manufacturing an embolizing agent precursor

ABSTRACT

Disclosed herein are methods relating to manufacturing an embolizing agent precursor. Manufacture of the embolizing agent precursor may involve mixing a first component contained within a first container with a second component contained within a second container, the first component including a plurality of negatively charged gaseous components and a first stabilizer, the second component comprising a plurality of positively charged oil components, a second stabilizer, and a cationic surfactant. Further steps may include mixing the first component with the second component such that the first and second component are held together as a single agglomerated entity.

FIELD OF THE INVENTION

The present invention relates to ultrasound (US) mediated delivery oftherapeutic agents, such as the delivery of a drug, gene, nanoparticleor radioisotope, using a bi-phasic microparticle system comprising gasmicrobubbles, emulsion microdroplets and clusters thereof. Thus, thepresent invention relates to a cluster composition and a pharmaceuticalcomposition, and their use for delivery of therapeutic agents and as acontrast agent for ultrasound imaging. It further relates to methods fordelivering such therapeutic agents and to the use of said compositions.

BACKGROUND OF THE INVENTION

A prerequisite for a successful medicinal therapy is that the drugreaches its target pathology and that the toxicity towards healthytissue is limited. However, a number of drugs display a low therapeuticindex severely limiting their clinical utility. Over the last fewdecades, the pharmaceutical industry has spent considerable resources intrying to solve this dilemma with various approaches fortargeted/localized drug delivery applying e.g. nanoparticle, microbubble or liposome platforms. By localized delivery of the drug to thepathology or organ in question the systemic exposure is minimized(reducing toxicity), and the local concentration, and thereby theefficacy, is increased.

Despite significant efforts, controlled drug delivery remainsessentially unresolved in clinical medicine. In recent years, researchand development have paid particular attention to externally activateddrug delivery systems. Heat, light, ultrasound, electric and magneticfields have been used as external energy sources for activating a drugformulation system in-vivo for release and delivery of drugs at targetedlocations within the body. For a recent review, see Timko et al [B.P.Timko et al, Remotely triggerable drug delivery systems, Adv. Mater. 22(2010) 4925-4943].

Over the past two decades, there has been growing interest in drugdelivery using ultrasound. For a recent review see Castle et al [Castleet al, Am J Physiol Heart Circ Physiol Feb. 1, 2013 304:H350-H357]. Manyapproaches are based on the use of microbubbles similar to those used asultrasound contrast agents for medical imaging applications, for releaseof incorporated or attached drugs and/or for enhanced uptake ofsystemically (co-)administered drugs.

Microbubbles have the potential of altering the structure of tissue andcell membranes via mechanisms such as sonoporation, hence enhancingextravasation of the released or co-administered drug to the targetedtissue. Application of ultrasound oscillates microbubbles present in themicrocirculation and induce well-established mechanisms that increasethe local permeability of the vasculature, allowing drugs to diffuse atan increased rate into the tissue space [O′Neill, BE and Li, KC, Int. J.Hyperthermia, September 2008; 24(6): 506-520]. Several mechanisms areknown to induce such bio effects, including sonoporation and endocytosisfor intracellular delivery, disruption of the endothelium and/orincreased opening/(reversible) modification of the fenestration pores oralteration of the vascular endothelium, or other mechanisms of enhancedtransport and diffusion such as radiation force and microstreaming. Therelative importance and exact nature of mechanisms and relation toultrasound dosimetry require further research and elucidation. Recentwork has also been motivated to address the issues of drug deliveryacross the blood brain barrier, and delivery to solid tumours. The bloodbrain barrier is characterised by tight vascular endothelial junctionsthat inhibits the passage of larger molecules to the tissue space.Tumour vasculature is generally more ‘leaky’ but suffers from higherinterstitial fluid and oncotic pressure that can impede passage of drugthroughout the tumour bulk. Uptake of established chemotherapeutics canbe highly variable depending on tumour type and such uptake differencesmay contribute to the variable nature of the therapeutic effect.Although microbubble mediated delivery mechanisms have been clearlydemonstrated in vivo, there are related bio-effects that raise safetyissues for the approach. To all likelihood, microbubble cavitationmechanisms are involved and in particular micro-haemorrhage andirreversible vascular damage has been observed. For techniques thataddress application to the blood brain barrier, there are also issuesrelated to delivering sufficient ultrasound energy to the pathologicalarea of interest, particularly if the overlying skull bone remainsintact and is not removed.

The most basic form of ultrasound/microbubble mediated drug delivery isadministration of a microbubble formulation together with a systemicallyadministered drug such as a enhanced uptake to the targeted pathology.An example of such an approach has recently entered clinical trials[Kotopoulis et al, Med Phys., 40(7) (2013)], where the commercial UScontrast agent Sonovue (Bracco Spa.) is co-administered with Gemcitabinefollowed by US irradiation for treatment of pancreatic cancer.

In addition to the co-administration approach, there are three generalclasses of microbubble technologies explored for drug delivery [Geers etal, Journal of Controlled Release 164 (2012) 248-255]: (1) drug loadedmicrobubbles; (2) in situ formed microbubbles from nanodroplets; and (3)targeted microbubbles (e.g. microbubbles with ligands attached fortargeting to cell surface receptors). Over the years, however, it hasbeen recognized that all these approaches have fundamental limitations,which have effectively hindered a transition to clinical practice.Perhaps the most limiting is the amount of drug that can be incorporatedinto microbubble systems. The thin, stabilizing shell or membranecarries a limited volume available for drug loading, and it has beenestimated that litres of a regular US contrast agent will be required inorder to obtain a therapeutic dose for common chemotherapeutic drugs[Geers et al, Journal of Controlled Release 164 (2012) 248-255]. Inaddition, for attachment and/or incorporation of the drug load into themicrobubble systems, chemical modification of the drug may be required,with potential changes to biological activity.

Microbubbles are also free flow blood tracers, and as soon as they havebeen triggered to release their payload, the drug will immediately startto wash out with the blood flow. As noted, a more basic microbubbleapproach includes co-injection with a regular drug formulation. Althoughsuch a concept does not have the limitation of low drug load, themicrobubbles are micron-sized and as such remain in the vascular space,and consequently bio-effects such as sonoporation for facilitatingenhanced uptake will be restricted to the vascular endothelium. Inaddition, the microbubbles are small and normally not in contact withthe vessel walls, limiting the magnitude and range of the bio-effectsfor enhanced drug uptake.

A different approach utilize nano emulsion technologies. Acousticmicrodroplet vaporisation (ADV) techniques have been described for anumber of applications including drug delivery and embolotherapy[Stanley, S. et al., Microcirculation 19: 501-509 (2012), Reznik, N.,Phys. Med. Biol. 57 (2012) 7205-7217]. These microdroplets are smallenough (typically less than 200 nm) to extravasate the (tumour) bloodvessels via the enhanced permeability and retention (EPR) effect andhave the advantage of overcoming the short circulation time of drugloaded microbubbles. They may be induced to evaporate (liquid to gasphase transition, i.e. phase shift) in-vivo by appliance of ultrasoundirradiation. However, these nano microdroplets have the disadvantage ofrequiring very high acoustic power to facilitate a phase shift of theoil in the nano microdroplets to a gaseous phase and providing a gasmicrobubble. Within medical US, acoustic power is normally described by“the Mechanical Index” (MI). This parameter is defined as the peaknegative pressure in the ultrasound field (PNP), de-rated by 0.3dB/cm/MHz divided be the square root of the centre frequency of theultrasound field in MHz ( F_(c) ) [American Institute of Ultrasound inMedicine. Acoustic Output Measurement Standard for Diagnostic UltrasoundEquipment. 1st ed. 2nd ed. Laurel, MD: American Institute of Ultrasoundin Medicine; 1998, 2003].

$MI = \frac{PNP}{\sqrt{F_{c}}}.$

Regulatory requirements during medical US imaging are to use a MI lessthan 1.9. During US imaging with microbubble contrast agents, an MIbelow 0.7 is recommended to avoid detrimental bio-effects such asmicro-haemorrhage and irreversible vascular damage and using an MI below0.4 is considered “best practise” [Miller et al, J Ultrasound Med 2008;27:611-632, AIUM Consensus Report on Potential Bioeffects of DiagnosticUltrasound].

For ADV, typically, a MI > 4 at 3.5 MHz needs to be applied tofacilitate an efficacious phase shift of the oil, powers which are wellabove the regulatory requirements to medical ultrasound imaging (MI <1.9) and far above the recommended MI of less than 0.7, and carrysignificant related bio-effects that raise safety issues for theapproach, particularly micro-haemorrhage and irreversible vasculardamage. In addition, they tend to re-condense to microdroplets almostimmediately after the phase shift event; hence, potential sonoporationmechanisms for improvement of drug bioavailability are limited.

To some extent, targeted microbubbles may improve the specificity fordrug delivery to the targeted pathology and/or the extent of thesonoporation bio effects, but technology is complex and again, limitedsuccess and transition to clinical use has emerged from these efforts.

Alternatives to the approaches reviewed above, recognized as prior artto the current invention have also been suggested. Note that in thebrief review of prior art presented below, the terminology used withinthese patents are kept. This may deviate slightly from the definitionsused in the current invention and detailed on pages 6 to 7.

WO 98/17324, “Improvements in or relating to contrast agents”, proposesa combined preparation comprising 1) a microbubble composition and 2) a“diffusible composition”, e.g. in the form of an oil in water emulsion,capable of diffusion in vivo into the microbubble composition,transiently increasing its size. In brief, this patent teaches thatapplication of ultrasound, after co-administration of these twocompositions, activates the bi-phasic (gas/liquid) system with anensuing liquid-to-gas phase shift of the diffusible component andgeneration of large phase shift bubbles that transiently traps in themicrovasculature, and hence could be used as a deposit tracer, UScontrast agent. For the diffusible composition, the patent teaches theuse of oils that are essentially insoluble and immiscible in water andwhich exist as gasses or display a substantial vapour pressure at bodytemperature. WO 98/17324 notes the possibility of using the proposedsystem for drug delivery by attaching a therapeutic component to themicrobubble composition. The patent also notes the possibility of mixingthe two compositions prior to simultaneous administration, but statesthat the mixture would then typically need to be stored at elevatedpressures or reduced temperatures in order to avoid spontaneous growthof the microbubbles prior to administration.

WO 98/51284, “Novel acoustically active drug delivery systems”, proposesa therapeutic delivery system comprising a microbubble wherein thebubble comprises an oil, a surfactant and a therapeutic agent dissolvedin the oil layer. In brief, this patent teaches that application ofultrasound, after administration, will disrupt the microbubbles andinduce a localized release of their drug load. It also teaches thepreferred use of oils with a melting point between -20 to 42° C. The oilcomponent is presented as a carrier (solvent) for the therapeutic agentand with the melting point rage in question, these oils will not serveas a “diffusible component” as taught in WO 98/17324.

WO 99/53963, “Improvements in or relating to contrast agents” buildsfurther on the invention detailed in WO 98/17324. In brief, this patentteaches that the efficacy of preparations of the type disclosed in WO98/17324 may be substantially enhanced if the two components areformulated such that the microbubbles and the diffusible component haveaffinity for each other, e.g. as a result of attractive electrostaticforces. WO 99/53963 also notes the possibility of using the proposedsystem for drug delivery by attaching a therapeutic component to themicrobubble composition. As in WO 98/17324 the patent notes thepossibility of mixing the two compositions prior to simultaneousadministration, but states that the mixture would then typically need tobe stored at elevated pressures or reduced temperatures in order toavoid spontaneous growth of the microbubbles prior to administration.

Both WO 98/17324 and WO 99/53963 consistently describe administration(simultaneous, separate or sequential) of two distinct compositions; adisperse gas (microbubble) composition and a diffusible composition(microdroplet emulsion).

Neither WO 98/17324 nor WO 99/53963 describe loading a therapeutic agentto the diffusible (microdroplet emulsion) composition.

Neither WO 98/17324 nor WO 99/53963 describe the use of US insonationafter activation of the phase shift event to facilitate extravasation ofdrug from the vascular compartment to target tissue.

Whereas the prior art cited above represents potential improvements overcurrently explored technologies utilising microbubbles as such, therehas been no transition to clinical practise and there is still a strongneed for an improved method for ultrasound mediated drug delivery.

DEFINITIONS

The term ‘microbubble’ or ‘regular, contrast microbubble’ is used inthis text to describe microbubbles with a diameter in the range from 0.2to 10 microns, typically with a mean diameter between 2 to 3 µm‘Regular, contrast microbubbles’ include commercially available agentssuch as Sonazoid (GE Healthcare), Optison (GE Healthcare), Sonovue(Bracco Spa.), Definity (Lantheus Medical Imagin), Micromarker(VisualSonics Inc.) and Polyson L (Miltenyi Biotec GmbH).

The term HEPS/PFB microbubble is used in this text to describe themicrobubbles formed by reconstituting the 1^(st) component (seeExample 1) with 2 mL of water.

The terms ‘phase shift bubbles’, ‘large, phase shift bubbles,’ ‘large,activated bubbles’ and ‘activated bubbles’ in this text is used todescribe the large (> 10 µm) bubbles that forms after US inducedactivation of the cluster composition.

The term ‘microdroplet’ is used in this text to describe emulsionmicrodroplets with a diameter in the range from 0.2 to 10 microns.

The term ‘emulsion’ is used in this text to describe an aqueoussuspension or dispersion of microbubbles.

The term ‘surfactant’ is used in this text for chemical compounds thatlower the surface tension between two liquids, e.g. used a stabiliser ina dispersion of microdroplets, or a gas and a liquid, e.g. used as astabiliser in a dispersion of microbubbles.

The term ‘nanoparticle’ is used in this text to describe particles withlinear dimensions less than 200 nm.

‘Insonation’ or ‘US irradiation’ are terms used to describe exposure to,or treatment with, ultrasound.

The term ‘deposit tracer’ is used in this text is used in relation tothe activated phase shift bubbles, in the sense that the temporarymechanical trapping of the large bubbles in the microcirculation impliesthat the regional deposition of phase shift bubbles in the tissue willreflect the amount of blood that flowed through the microcirculation ofthe tissue at the time of activated bubble deposition. Thus, the numberof trapped ‘deposited’ phase shift bubbles will be linearly dependent onthe tissue perfusion at the time of deposition.

The term ‘phase shift (process)’ is used in this text to describe thephase transition from the liquid to gaseous states of matter.Specifically the transition (process) of the change of state from liquidto gas of the oil component of the microdroplets of the clustercomposition.

The term ‘bi-phasic’ refers to a system comprising of two phases ofstate, specifically liquid and gaseous states, such as the microbubble(gas) and microdroplet (liquid) components of the cluster composition.

In this text the terms “therapy delivery / therapeutic agent(s)” and“drug delivery / drug(s)” are both understood to include the delivery ofdrug molecules, nanoparticles and nanoparticle delivery systems, genes,and radioisotopes.

The term ‘1^(st) component’ is used in this text to describe thedispersed gas (microbubble) component.

The term ‘2^(nd) component’ is used in this text to describe thedispersed oil phase (microdroplet) component comprising a diffusiblecomponent.

The term ‘cluster composition’ is used in this text to describecomposition resulting from a combination of the 1^(st) (microbubble)component and the 2^(nd) (microdroplet) component.

The term “diffusible component” is used in this text to describe achemical component of the oil phase of the 2^(nd) component that iscapable of diffusion in vivo into the microbubbles in the 1^(st)component of, transiently increasing its size.

The term ‘loading capacity’ is used in this text to describe the amount(capacity) of the drug that can be incorporated into the drug deliveryvehicle.

The term “pharmaceutical composition” used in this text has itsconventional meaning, and in particular are in a form suitable formammalian administration, especially via parenteral injection. By thephrase “in a form suitable for mammalian administration” is meant acomposition that is sterile, pyrogen-free, lacks compounds which produceexcessive toxic or adverse effects, and is formulated at a biocompatiblepH (approximately pH 4.0 to 10.5). Such a composition is formulated sothat precipitation does not occur on contact with biological fluids(e.g. blood), contain only biologically compatible excipients, and ispreferably isotonic.

The term ‘Sonometry (system)’ in this text refers to a measurementsystem to size and count activated phase shift bubbles dynamically usingan acoustic technique.

The term ‘Reactivity’ is used in this text to describe the ability ofthe microbubbles in the 1^(st) component and the microdroplets in the2^(nd) component to form microbubble/microdroplet clusters upon mixing.

The terms “microbubble/microdroplet cluster” or “cluster” in this textrefers to groups of microbubbles and microdroplets permanently heldtogether by electrostatic attractive forces, in a single particle,agglomerated entity.

The term ‘clustering’ in this text refers to the process wheremicrobubbles in the 1^(st) component and microdroplets of the 2^(nd)component forms clusters.

The term ‘activation’ in this text refers to the induction of a phaseshift of microbubble/microdroplet clusters by US irradiation.

ABBREVIATIONS

-   H_(d): Hansen distance.-   ADV: Acoustic microdroplet vaporisation.-   ANOVA: analysis of variance-   a.u.: arbitrary units-   b.p:. boiling point.-   b.w.: body weight-   C: circularity.-   C1: 1^(st) component-   C2: 2^(nd) component-   dCldFEt: dichlorodifluoroethane.-   CltFPr: chlorotrifluropropane.-   COO: continuous cardiac output.-   CV: cross validation.-   dB: decibel.-   dClMe: dichloromethane.-   DiR: near infrared fluorescent dye.-   DP: cluster composition or pharmaceutical composition-   DSPC: 1,2 Distrearoyl-sn-glycerol-3-phosphocholine.-   e.g.: for example-   FPIA: Flow Particle Image Analysis.-   HEPS: hydrogenated egg sodium-phosphatidyl serine.-   HIFU: high intensity focused ultrasound.-   i.v: intravenous.-   LogP: logarithm (to the base 10) of the (octanol/water) partition    coefficient, a measure of lipophilicity.-   LogS: logarithm (to the base 10) of the aqueous solubility in gr/100    mL-   M: molar-   MI: MI.-   NA: not applicable.-   NR: nile red fluorescent dye.-   PBS: phosphate buffered saline.-   PCA: principal component analysis-   PFB: perflurobutane.-   pFMCP: perfluoromethyl-cyclopentane-   PLSR: partial least squares regression.-   QC: quality control.-   R: Reactivity of the cluster composition.-   SA: stearlyamine-   tClMe: trichloromethane.-   TIC: time intensity curve-   TRIS: 2-Amino-2-hydroxymethyl-propane-1,3-diol.-   US: Ultrasound.-   v.p:. vapour pressure.-   v/v: volume per volume.-   ~: approximate.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that drug delivery can beachieved by the use of a two component, bi-phasicmicrobubble/microdroplet formulation system where microbubbles in afirst component, via electrostatic attraction, are physically attachedto micron sized emulsion microdroplets in a second component prior toadministration. Contrary to the teachings in WO 99/53963 we have foundthat mixing the first component with the second component prior toadministration is a pre-requisite for the efficient formation of suchmicrobubble/microdroplet clusters and that the cluster composition canbe stable at ambient conditions. The clusters are readily activatedin-vivo with low power ultrasound (i.e. with an MI of less than 1.9,preferably less than 0.7 and most preferably less than 0.4), whichinduce a liquid-to-gas transition (phase shift) of the diffusiblecomponent. A therapeutic agent may be added to the microdroplet oilphase and/or co-administered as a regular drug formulation. The large,activated bubbles are temporarily retained in the microvasculature andmay be utilized to facilitate drug uptake to target tissue by furtherapplication of ultrasound.

The drug delivery technology of the present invention differs markedlyfrom the existing ultrasound mediated drug delivery technologies andprior art outlined above. Main improvements/novelty elements vs.standard microbubble approaches are:

-   A marked increase in drug loading capacity where the entire volume    of a large (micron sized) emulsion microdroplet can be utilized for    drug payload vs. only the thin stabilizing membrane of the    microbubble component for the prior art approaches.-   The size of the activated phase shift bubbles being approx. 10 times    larger than typical microbubbles: trapping of the activated bubbles    in the microvasculature: transient stopping of blood flow, avoiding    a rapid wash out of the drug: close contact between the activated    bubbles and the endothelium: orders of magnitude larger bio-effects    during post activation US treatment, avoiding cavitation mechanisms.

Main improvements/novelty elements vs. acoustic microdropletvaporization (ADV) approaches are:

-   Significantly lower acoustic power is required to produce the phase    shift event; typically an MI of 0.2 to 0.4 is required for the    current invention vs. typically > 4 for ADV.-   Significantly longer lifetime of activated bubbles, no rapid    recondensing;-   Activation in the vascular compartment vs. activation in tissue.-   The size of the activated phase shift bubbles being approx. 10 times    larger than typical microbubbles from ADV: trapping of the activated    bubbles, avoiding a rapid wash out of the drug: close contact    between the activated bubbles and the endothelium: orders of    magnitude larger bio-effects during post activation US treatment,    avoiding cavitation mechanisms.

Main improvements/novelty elements vs. phase shift approaches describedin WO 98/1732 and WO 99/53963 are:

-   The formation of stable microbubble/microdroplet clusters in a    single, combined cluster composition prior to administration, with    an ensuing ~ 10 fold increase in deposit capacity.-   A marked increase in drug loading capacity where the entire volume    of a large (micron sized) emulsion microdroplet can be utilized for    drug payload vs. only the thin stabilizing membrane of the    microbubble component.-   The continued application of acoustic energy, post activation, to    facilitate drug delivery.

A cluster composition, a pharmaceutical composition for delivery ofdrugs and a method for delivery has now been identified that uses phaseshift technology of the current invention to generate large phase shiftbubbles in vivo from an administered composition containingmicrobubble/microdroplet clusters, and which facilitates delivery ofassociated and/or pre-, and/or co- and/or post administered therapeuticagent(s).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides a cluster composition thatcomprises a suspension of clusters in an aqueous biocompatible medium,where said clusters have a diameter in the range of 1 to 10 µm, and acircularity < 0.9 and comprise:

-   (i) a first component which comprises a gas microbubble and first    stabiliser to stabilise said microbubble; and-   (ii) a second component which comprises a microdroplet comprising an    oil phase and second stabiliser to stabilise said microdroplet,    where the oil comprises a diffusible component capable of diffusing    into said gas microbubble so as to at least transiently increase the    size thereof, where said second component optionally further    comprises a first therapeutic agent;-   where the microbubbles and microdroplets of said first and second    components have opposite surface charges and form said clusters via    attractive electrostatic interactions.

The cluster composition, i.e. the combination of the first and secondcomponents, comprises clusters of gas microbubbles and oilmicrodroplets, i.e. is a suspension or dispersion of individualmicrobubbles and microdroplets together with stablemicrobubble/microdroplet clusters. The cluster composition is intendedfor administration (e.g. intravenously) to a mammalian subject.Analytical methodologies for quantitative detection and characterisationof said clusters are described in Example 1.

In this text, the term “clusters” refers to groups of microbubbles andmicrodroplets permanently held together by electrostatic attractiveforces, in a single particle, agglomerated entity. The content and sizeof the clusters in the cluster composition is essentially stable oversome time (e.g. > 1h) after combining the first and second components invitro, i.e. they do not spontaneously disintegrate, form largeraggregates or activates (phase shifts) spontaneously, and areessentially stable over some time after dilution, even during continuedagitation. It is hence possible to detect and characterize the clustersin the cluster composition with various analytical techniques thatrequire dilution and/or agitation.

The clusters typically contain at least one microbubble and onemicrodroplet, typically 2-50 individual microbubbles/microdroplets, aretypically 1 to 10 µm in diameter and can hence flow freely in thevasculature. They are further characterized and separated fromindividual microbubbles and microdroplets by a circularity parameter.The circularity of a two-dimensional form (e.g. a projection of amicrobubble, microdroplet or microbubble/microdroplet cluster) is theratio of the perimeter of a circle with the same area as the form,divided by the actual perimeter of the form. Several mathematicalalgorithms for calculating and reporting of circularity are used.Normally, in order to provide a response that is sensitive to smalldeviations from a perfect circular form, the squared circularity (alsotermed ‘high sensitivity circularity’) is calculated and reported. Thisterm (C) has its conventional meaning in the field of image analysis[Wojnar, L. et al., Practical Guide to Image Analysis, ASMInternational, 2000, p 157-160] and is a mathematical definition ofroundness in two dimensions expressed as:

C = 4πA/P²

Where A is the two dimensional, projection area of the form and P is thetwo dimensional, projection perimeter of the form. Accordingly, aperfect circle (i.e. a two dimensional projection of a sphericalmicrobubble or microdroplet) has a theoretical circularity value of 1,and any other geometrical form (e.g. projection of a cluster) has acircularity of less than 1. In the current text, circularity (C) asdefined above is used.

As noted, contrary to the teachings in WO 99/53963 the present inventorshave found that mixing the first component with the second componentprior to administration is a pre-requisite for the efficient formationof such microbubble/microdroplet clusters and that the clustercomposition can be stable at ambient conditions. In addition, theinventors have found that it is the clustered form of the microbubblesand microdroplets in the cluster composition that enables an efficaciousactivation (phase shift) and deposition of the activated bubbles in-vivoin the vasculature. As detailed in Examples 1 and 2, we have also foundthat engineering various aspects of the clusters of the invention, theirconcentration and size in particular; a ~ 10-fold increase in depositand drug delivery capacity can be achieved, compared to simultaneousco-administration of the two separate microbubble and microdropletcomponents.

According to the invention, clusters in the size range 1-10 µm definedby a circularity of < 0.9 are considered particularly useful, asdemonstrated in Examples 2 and 5-1. Clusters in this size range arefree-flowing in the vasculature before activation, they are readilyactivated by US irradiation and they produce activated bubbles that arelarge enough to deposit and lodge temporarily in the microvasculature.

It has been found that the presence of the microbubbles in the clusterspermits efficient energy transfer of ultrasound energy in the diagnosticfrequency range (1-10 MHz), i.e. activation, and allows vaporisation(phase shift) of the emulsion microdroplets at low MI (under 1.9 andpreferably under 0.7 and more preferably under 0.4) and diffusion of thevaporized liquid into the microbubbles and/or fusion between the vapourbubble and the microbubble. The activated bubble then expands furtherfrom the inwards diffusion of matrix gases (e.g. blood gases) to reach adiameter of more than 10 µm, preferably more than 20 µm. The exactmechanisms involved during activation of the clusters and generation ofphase shift bubbles needs further research and elucidation.

It has further been found that the formation of theses clusters is aprerequisite for an efficient phase shift event and that their numberand size characteristics are strongly related to the efficacy of thecomposition, i.e. its ability to form large, activated (i.e. phasedshifted) bubbles in-vivo. The number and size characteristics can becontrolled through various formulation parameters such as, but notlimited to; the strength of the attractive forces between themicrobubbles in the first component and the microdroplets in the secondcomponent (e.g. the difference in surface charge between themicrobubbles and microdroplets): the size distribution of microbubblesand microdroplets: the ratio between microbubbles and microdroplets: andthe composition of the aqueous matrix (e.g. buffer concentration, ionicstrength) (see Examples 1 and 2).

The size of the activated bubbles can be engineered by varying the sizedistribution of the microdroplets in the emulsion and the sizecharacteristics of the clusters (see Example 1). The clusters areactivated to produce large bubbles by application of external ultrasoundenergy, such as from a clinical ultrasound imaging system, under imagingcontrol. The large phase shift bubbles produced are typically of adiameter of 10 µm or more (see Examples 1, 2, 3 and 4). Low MI energylevels, which are well within the diagnostic imaging exposure limits (MI< 1.9), are sufficient to activate the clusters which make thetechnology significantly different from the other phase transitiontechnologies available (e.g. ADV).

Due to the large size of the activated bubbles, they temporarily lodgein the microvasculature and can be spatially localised in a tissue ororgan of interest by spatially localised deposition of the ultrasoundenergy to activate the clusters (see Examples 4 and 7). The large,activated bubbles produced (10 µm or more in diameter) have acousticresonances at low ultrasound frequency (1 MHz or less). It has beenfound that the application of low frequency ultrasound, close to theresonance frequencies of the large, activated bubbles (i.e. frequencycomponents in the range 0.05 to 2 MHz, preferably in the range 0.1 to1.5 MHz, most preferably in the range of 0.2 to 1 MHz), can be used toproduce mechanical and/or thermal bio-effect mechanisms to increase thelocal permeability of the vasculature and/or sonoporation and/orendocytosis and hence increase delivery and retention of drugs (seeExample 8).

It will be appreciated that the delivery mechanisms during this USirradiation will be substantially different from those induced whenapplying US to regular, free flowing microbubbles such as contrastagents for US imaging. Whereas the large phase shift microbubbles areentrapped in a segment of the vessels and the microbubble surface is inclose contact with the endothelium, micron sized microbubbles arefree-flowing and (on average) relatively far from the vessel wall (seeExample 4). In addition, the volume of an activated bubble from thecurrent invention is typically 1000 times that of a regular microbubble.At equal MI, insonated at a frequency close to resonance for both bubbletypes (0.5 MHz for phase shift microbubbles and 5 MHz for regularcontrast agent microbubbles) it has been shown that the absolute volumedisplacement during oscillations are almost three orders of magnitudelarger with the phase shift bubbles than with a regular contrastmicrobubble. Hence, insonation of phase shift bubbles will producecompletely different levels of bio-mechanical effects, withsignificantly larger effect size and penetration depth than duringinsonation of regular contrast microbubbles. The bio-effects observedwith free-flowing, regular contrast microbubbles are likely dependentupon cavitation mechanisms, with ensuing safety concerns such asmicro-haemorrhage and irreversible vascular damage. The larger phaseshift bubbles can be oscillated in a softer manner (lower MI, e.g. <0.4), avoiding cavitation mechanisms, but still inducing sufficientmechanical work to enhance the uptake of drug from the vasculature andinto the target tissue (see Example 8).

The trapping of the large phase shift bubbles will also act as a deposittracer. This further allows quantification of the number of activatedclusters and perfusion of the tissue, and allows contrast agent imagingof the tissue vasculature to identify the spatial extent of thepathology to be treated (see Example 7).

The cluster composition, i.e. comprising the combination of the firstand second components, is comprised of a bi-phasic micro particle systemengineered to cluster and phase shift in a controlled manner. Drug maybe incorporated into low boiling point, micron sized oil microdropletsof the second component, which are stabilised e.g. with a positivelycharged phospholipid membrane. Before administration, the drug loadedoil microdroplets of the second component are mixed with micron sizedgas microbubbles in the first component. Such gas microbubbles mayconsist of, for example but not limited to, a low solubilityperfluorocarbon gas core stabilised with a negatively chargedphospholipid membrane.

When exposed to ultrasound (standard medical imaging frequency andintensity) at the targeted pathology, the microbubble transfers acousticenergy to the attached oil microdroplets and acts as a ‘seed’ for theoil to undergo a liquid-to-gas phase shift (vaporisation). During thisprocess the drug load is released from the oil phase or expressed at thesurface of the activated bubble. The resulting bubble undergoes aninitial rapid expansion due to vaporisation of the oil, followed by aslower expansion due to inward diffusion of blood gases, to at least 10µm diameter or more, preferably at least 20 µm diameter or more, andtemporarily blocks the microcirculation (met arteriole and capillarynetwork), transiently stopping blood flow for approximately 1 minute ormore, preferably 2-3 minutes or more, most preferably 3-6 minutes ormore, keeping the released or expressed drug at high concentration andclose proximity to the target pathology (see Examples 4 and 7).

Alternatively, if the therapeutic agent is not incorporated into the oilphase of the emulsion microdroplet, the therapeutic agent can beseparately administered, such as being co-administered orpre-administered or post-administered with the cluster composition. Inthis case, the therapeutic agent can be administered in any convenientfor including, but not limited to, injectable or oral forms, e.g. as aregular drug formulation, e.g. Taxol, Gemzar or other marketedchemotherapeutics. Alternatively, a therapeutic agent may be includedboth in the oil phase and administered as a separate composition.Activation of the phase shift technology produces large phase shiftbubbles which are trapped at the site of interest temporarily stoppingblood flow which contains the separately administered therapeutic agent.Further application of ultrasound after trapping facilitatesextravasation of the drug to the targeted tissue.

The first component of the present invention contain microbubbles thatare similar to conventional ultrasound contrast agents that are on themarket and approved for use for several clinical applications such asSonazoid, Optison, Definity or Sonovue, or similar agents used forpre-clinical application such as Micromarker and Polyson L. The firstcomponent is an injectable aqueous medium comprising dispersed gas andmaterial to stabilise said gas. Any biocompatible gas may be present inthe gas dispersion, the term “gas” as used herein including anysubstances (including mixtures) at least partially, e.g. substantiallyor completely in gaseous (including vapour) form at the normal humanbody temperature of 37° C. The gas may thus, for example, comprise air;nitrogen; oxygen; carbon dioxide; hydrogen; an inert gas such as helium,argon, xenon or krypton; a sulphur fluoride such as sulphurhexafluoride, disulphur decafluoride or trifluoromethylsulphurpentafluoride; selenium hexafluoride; an optionally halogenated silanesuch as methylsilane or dimethylsilane; a low molecular weighthydrocarbon (e.g. containing up to 7 carbon atoms), for example analkane such as methane, ethane, a propane, a butane or a pentane, acycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkenesuch as ethylene, propene, propadiene or a butene, or an alkyne such asacetylene or propyne; an ether such as dimethyl ether; a ketone; anester; a halogenated low molecular weight hydrocarbon (e.g. containingup to 7 carbon atoms); or a mixture of any of the foregoing.Advantageously at least some of the halogen atoms in halogenated gasesare fluorine atoms; thus biocompatible halogenated hydrocarbon gasesmay, for example, be selected from bromochlorodifluoromethane,chlorodifluoromethane, dichlorodifluoromethane, bromotrifluoromethane,chlorotrifluoromethane, chloropentafluoroethane,dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene,ethylfluoride, 1,1-difluoroethane and perfluorocarbons. Representativeperfluorocarbons include perfluoroalkanes such as perfluoromethane,perfluoroethane, perfluoropropanes, perfluorobutanes (e.g.perfluoro-n-butane, optionally in admixture with other isomers such asperfluoro-iso-butane), perfluoropentanes, perfluorohexanes orperfluoroheptanes; perfluoroalkenes such as perfluoropropene,perfluorobutenes (e.g. perfluorobut-2-ene), perfluorobutadiene,perfluoropentenes (e.g. perfluoropent-1-ene) orperfluoro-4-methylpent-2-ene; perfluoroalkynes such asperfluorobut-2-yne; and perfluorocycloalkanes such asperfluorocyclobutane, perfluoromethylcyclobutane,perfluorodimethylcyclobutanes, perfluorotrimethyl-cyclobutanes,perfluorocyclopentane, perfluoromethyl-cyclopentane,perfluorodimethylcyclopentanes, perfluorocyclohexane,perfluoromethylcyclohexane or perfluorocycloheptane. Other halogenatedgases include methyl chloride, fluorinated (e.g. perfluorinated) ketonessuch as perfluoroacetone and fluorinated (e.g. perfluorinated) etherssuch as perfluorodiethyl ether.

The use of perfluorinated gases, for example sulphur hexafluoride andperfluorocarbons such as perfluoropropane, perfluorobutanes,perfluoropentanes and perfluorohexanes, are particularly advantageous inview of the recognised high stability in the bloodstream of microbubblescontaining such gases. Other gases with physicochemical characteristicswhich cause them to form highly stable microbubbles in the bloodstreammay likewise be useful. Most preferably, the dispersed gas comprisesulphur hexafluoride, perfluoropropane, perfluorobutane,perfluoropentane, perflurohexane, nitrogen, air or a mix thereof.

The dispersed gas may be in any convenient form, for example using anyappropriate gas-containing ultrasound contrast agent formulation as thegas-containing component such as Sonazoid, Optison, Sonovue or Definityor pre-clinical agents such as Micromarker or PolySon L. The firstcomponent will also contain material in order to stabilise themicrobubble dispersion, in this text termed ‘first stabiliser’.Representative examples of such formulations include microbubbles of gasstabilised (e.g. at least partially encapsulated) by a first stabilisersuch as a coalescence-resistant surface membrane (for example gelatin),a filmogenic protein (for example an albumin such as human serumalbumin), a polymer material (for example a synthetic biodegradablepolymer, an elastic interfacial synthetic polymer membrane, amicroparticulate biodegradable polyaldehyde, a microparticulateN-dicarboxylic acid derivative of a polyamino acid - polycyclic imide),a non-polymeric and non-polymerisable wall-forming material, or asurfactant (for example a polyoxyethylene-polyoxypropylene blockcopolymer surfactant such as a Pluronic, a polymer surfactant, or afilm-forming surfactant such as a phospholipid). Preferably, thedispersed gas is in the form of phospholipid-, protein- orpolymer-stabilised gas microbubbles. Particularly useful surfactantsinclude phospholipids comprising molecules with net overall negativecharge, such as naturally occurring (e.g. soya bean or egg yolkderived), semisynthetic (e.g. partially or fully hydrogenated) andsynthetic phosphatidylserines, phosphatidylglycerols,phosphatidylinositols, phosphatidic acids and/or cardiolipins.Alternatively the phospholipids applied for stabilization may carry andoverall neutral charge and be added a negative surfactant such as afatty acid, e.g. phosphatidylcholine added palmitic acid, or be a mix ofdifferently charged phospholipids, e.g. phosphatidylethanolamines and/orphosphatidylcholine and/or phosphatidic acid.

The microbubble size of the dispersed gas component intended forintravenous injection should preferably be less than 7 µm, morepreferably less than 5 µm and most preferably less than 3 µm in order tofacilitate unimpeded passage through the pulmonary system, even when ina microbubble/microdroplet cluster.

For the second component the “diffusible component” is suitably agas/vapour, volatile liquid, volatile solid or precursor thereof capableof gas generation, e.g. upon administration, the principal requirementbeing that the component should either have or be capable of generatinga sufficient gas or vapour pressure in vivo (e.g. at least 50 torr andpreferably greater than 100 torr) so as to be capable of promotinginward diffusion of gas or vapour molecules into the dispersed gas. The‘diffusible component’ is preferably formulated as an emulsion (i.e. astabilised suspension) of microdroplets in an appropriate aqueousmedium, since in such systems the vapour pressure in the aqueous phaseof the diffusible component will be substantially equal to that of purecomponent material, even in very dilute emulsions.

The diffusible component in such microdroplets is advantageously aliquid at processing and storage temperature, which may for example beas low as -10° C. if the aqueous phase contains appropriate antifreezematerial, while being a gas or exhibiting a substantial vapour pressureat body temperature. Appropriate compounds may, for example, be selectedfrom the various lists of emulsifiable low boiling liquids given in thepatent WO-A-9416379, the contents of which are incorporated herein byreference. Specific examples of emulsifiable diffusible componentsinclude aliphatic ethers such as diethyl ether; polycyclic oils oralcohols such as menthol, camphor or eucalyptol; heterocyclic compoundssuch as furan or dioxane; aliphatic hydrocarbons, which may be saturatedor unsaturated and straight chained or branched, e.g. as in n-butane,n-pentane, 2-methylpropane, 2-methylbutane, 2,2-dimethylpropane,2,2-dimethylbutane, 2,3-dimethylbutane, 1-butene, 2-butene,2-methylpropene, 1,2-butadiene, 1,3-butadiene, 2-methyl-1-butene,2-methyl-2-butene, isoprene, 1-pentene, 1,3-pentadiene, 1,4-pentadiene,butenyne, 1-butyne, 2-butyne or 1,3-butadiyne; cycloaliphatichydrocarbons such as cyclobutane, cyclobutene, methylcyclopropane orcyclopentane; and halogenated low molecular weight hydrocarbons (e.g.containing up to 7 carbon atoms). Representative halogenatedhydrocarbons include dichloromethane, methyl bromide,1,2-dichloroethylene, 1,1-dichloroethane, 1-bromoethylene,1-chloroethylene, ethyl bromide, ethyl chloride, 1-chloropropene,3-chloropropene, 1-chloropropane, 2-chloropropane and t-butyl chloride.Advantageously at least some of the halogen atoms are fluorine atoms,for example as in dichlorofluoromethane, trichlorofluoromethane,1,2-dichloro-1,2-difluoroethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane,1,1,2-trichloro-1,2,2-trifluoroethane,2-bromo-2-chloro-1,1,1-trifluoroethane, 2-chloro-1,1,2-trifluoroethyldifluoromethyl ether, 1-chloro-2,2,2-trifluoroethyl difluoromethylether, partially fluorinated alkanes (e.g. pentafluoropropanes such as1H, 1H,3H-pentafluoropropane, hexafluorobutanes, nonafluorobutanes suchas 2H-nonafluoro-t-butane, and decafluoropentanes such as2H,3H-decafluoropentane), partially fluorinated alkenes (e.g.heptafluoropentenes such as 1H,1H,2H-heptafluoropent-1-ene, andnonafluorohexenes such as 1H,1H,2H-nonafluorohex-1-ene), fluorinatedethers (e.g. 2,2,3,3,3-pentafluoropropyl methyl ether or2,2,3,3,3-pentafluoropropyl difluoromethyl ether) and, more preferably,perfluorocarbons. Examples of perfluorocarbons include perfluoroalkanessuch as perfluorobutanes, perfluoropentanes, perfluorohexanes (e.g.perfluoro-2-methylpentane), perfluoroheptanes, perfluorooctanes,perfluorononanes and perfluorodecanes; perfluorocycloalkanes such asperfluorocyclobutane, perfluorodimethylcyclobutanes,perfluorocyclopentane and perfluoromethylcyclopentane; perfluoroalkenessuch as perfluorobutenes (e.g. perfluorobut-2-ene orperfluorobuta-1,3-diene), perfluoropentenes (e.g. perfluoropent-1-ene)and perfluorohexenes (e.g. perfluoro-2-methylpent-2-ene orperfluoro-4-methylpent-2-ene); perfluorocycloalkenes such asperfluorocyclopentene or perfluorocyclopentadiene; and perfluorinatedalcohols such as perfluoro-t-butanol.

Particularly useful in the current invention are diffusible componentswith an aqueous solubility below 1·10⁻⁴ M, more preferably below 1·10⁻⁵M (see Example 5-3). It should be noted, however, that if a mixture ofdiffusible components and/or co-solvents are used, a substantialfraction of the mixture may contain compounds with a higher watersolubility (see Example 5-4).

It will be appreciated that mixtures of two or more diffusiblecomponents may if desired be employed in accordance with the invention;references herein to “the diffusible component” are to be interpreted asincluding such mixtures. It will also be appreciated that drugs may beincorporated into the diffusible component(s) and that co-solvents,described in the text below, may also be used in order to increase thedrug loading capacity of the system.

The second component will also contain material in order to stabilisethe microdroplet dispersion, in this text termed ‘second stabiliser’.The second stabiliser may be the same as or different from anymaterials(s) used to stabilise the gas dispersion, e.g. a surfactant, apolymer or a protein. The nature of any such material may significantlyaffect factors such as the rate of growth of the dispersed gas phase. Ingeneral, a wide range of surfactants may be useful, for example selectedfrom the extensive lists given in EP-A-0727225, the contents of whichare incorporated herein by reference. Representative examples of usefulsurfactants include fatty acids (e.g. straight chain saturated orunsaturated fatty acids, for example containing 10-20 carbon atoms) andcarbohydrate and triglyceride esters thereof, phospholipids (e.g.lecithin), fluorine-containing phospholipids, proteins (e.g. albuminssuch as human serum albumin), polyethylene glycols, and polymer such asa block copolymer surfactants (e.g. polyoxyethylene-polyoxypropyleneblock copolymers such as Pluronics, extended polymers such asacyloxyacyl polyethylene glycols, for example polyethyleneglycol methylether 16-hexadecanoyloxy-hexadecanoate, e.g. wherein the polyethyleneglycol moiety has a molecular weight of 2300, 5000 or 10000), andfluorine-containing surfactants (e.g. as marketed under the trade namesZonyl and Fluorad, or as described in WO-A-9639197, the contents ofwhich are incorporated herein by reference). Particularly usefulsurfactants include phospholipids comprising molecules with overallneutral charge, e.g. distearoyl-sn-glycerol-phosphocoline.

It will be appreciated that, to facilitate attractive electrostaticinteractions to achieve clustering between the microbubbles in the firstcomponent and the emulsion microdroplets in the second component, theseshould be of opposite surface charge. Hence, if the microbubbles of thefirst component are negatively charged, the microdroplets of the secondcomponent should be positively charged, or vice versa. In order tofacilitate a suitable surface charge for the oil microdroplets acationic surfactant may be added to the stabilizing structure. A widerange of cationic substances may be used, for example at least somewhathydrophobic and/or substantially water-insoluble compounds having abasic nitrogen atom, e.g. primary, secondary or tertiary amines andalkaloids. A particularly useful cationic surfactant is stearylamine.

It will also be appreciated that the mixing of the first and secondcomponents can be achieved in various manners depended on the form ofthe components; e.g. mixing two fluid components, reconstitution of onecomponent in dry powder form with one component in fluid form, mixingtwo components in dry form prior to reconstitution with fluid (e.g.water for injection or buffer solution). Also, it will be appreciatedthat other components may influence the ability of the microbubbles andmicrodroplets to form clusters upon mixing including, but not limitedto; the level of surface charge of the microbubbles/microdroplets, theconcentration of the microbubbles/microdroplets in the two components,the size of the microbubbles/microdroplets, the composition andconcentration of ions, the composition and concentration of excipients(e.g. buffer or tonicity components) etc. (see Example 1). Suchcharacteristics of the components and the composition may also influencethe size and stability (both in-vitro and in-vivo) of the clustersgenerated and may be important factors influencing biological attributes(e.g. efficacy and safety profile). It is also appreciated that not allof the microbubbles/microdroplets in the cluster composition may bepresent in clustered form, but that a substantial fraction of themicrobubbles and/or microdroplets may be present together in a free(non-clustered) form together with a population ofmicrobubble/microdroplet clusters. In addition, the way the twocomponents are mixed may influence these aspects, including, but notlimited to; shear stress applied during homogenization (e.g. soft manualhomogenization or strong mechanical homogenization) and time range forhomogenization.

The microdroplet size of the dispersed diffusible component in emulsionsintended for intravenous injection should preferably be less than 7 µm,more preferably less than 5 µm, most preferably less than 3 µm, andgreater than 0.5 µm, more preferably greater than 1 µm in order tofacilitate unimpeded passage through the pulmonary system, but stillretain a volume that is sufficient for drug loading and activated bubbleretention in the microvasculature.

Growth of the dispersed gas phase in vivo may, for example, beaccompanied by expansion of any encapsulating material (where this hassufficient flexibility) and/or by abstraction of excess surfactant fromthe administered material to the growing gas-liquid interfaces. It isalso possible, however, that stretching of the encapsulating materialand/or interaction of the material with ultrasound may substantiallyincrease its porosity. Whereas such disruption of encapsulating materialhas hitherto in many cases been found to lead to rapid loss ofechogenicity through outward diffusion and dissolution of the gasthereby exposed, we have found that when using compositions inaccordance with the present invention, the exposed gas exhibitssubstantial stability. Whilst not wishing to be bound by theoreticalcalculations, we believe that the exposed gas, e.g. in the form ofliberated microbubbles, may be stabilised, e.g. against collapse of themicrobubbles, by a supersaturated environment generated by thediffusible component, which provides an inward pressure gradient tocounteract the outward diffusive tendency of the microbubble gas. Theexposed gas surface, by virtue of the substantial absence ofencapsulating material, may cause the activated bubbles to exhibitexceptionally favourable acoustic properties as evidenced by highbackscatter and low energy absorption (e.g. as expressed by highbackscatter: attenuation ratios) at typical diagnostic imagingfrequencies; this echogenic effect may continue for a significantperiod, even during continuing ultrasound irradiation.

The acoustic resonance of the microbubble component of the clusters iswithin the diagnostic frequency range (1-10 MHz). Activation of theclusters is readily obtained with standard diagnostic ultrasound imagingpulses used for example in conventional medical ultrasound abdominal andcardiac applications, at mid-range to low mechanical indices (MI below1.9 and preferably below 0.7 and more preferably below 0.4). Activationof the clusters to phase shift to produce larger (10 µm or more indiameter) phase shift bubbles can be achieved with a clinical imagingsystem to within millimetre spatial resolution by employing imagingpulses. Upon activation, the oil in the microdroplet vaporises,releasing the therapeutic agent (if included) to the surround fluid infree drug form, or as crystallised drug (in particulate form) orexpressed on/associated with the activated bubble surface. The activatedbubbles trap in the microvasculature, temporarily stopping blood flowand keeping the drug in the microvasculature at high concentration.Further application of ultrasound after trapping facilitates deliverymechanisms to increase the efficiency of drug delivery to the tissue.The clusters are not activated at low MI (below the cluster activationthreshold of approx. 0.1) allowing standard medical ultrasound contrastagent imaging to be performed, for example to identify tumour microvascular pathology without activation of the clusters. Activation undermedical ultrasound imaging control using the imaging pulses allowsspatially targeted activation of the clusters in the tissue region beinginterrogated by the ultrasound field. After activation, the large phaseshift bubbles produced are temporarily trapped in the microvasculaturedue to their size (10 µm or more in diameter). The resulting large phaseshift bubbles are approximately 1000 times the volume of the emulsionmicrodroplet vaporised (30 µm bubble diameter from a 3 µm diameter oilmicrodroplet). The scattering cross sections of these large phase shiftbubbles are orders of magnitude greater than the scattering crosssections of the micron sized microbubbles comprised in the clustersbefore activation. As a result, the large phase shift bubbles producecopious backscatter signal and are readily imaged in fundamental imagingmode with diagnostic imaging systems (see Examples 2 and 7). Themechanical resonance frequencies of the large phase shift bubbles arealso an order of magnitude lower (1 MHz or less) than the resonancefrequencies of the microbubbles comprised in the clusters beforeactivation. Application of acoustic fields commensurate with theresonance frequencies of the larger phase shift bubbles producesrelatively large radius oscillations at MI’s within the medicaldiagnostic range. Thus, low frequency (0.05 to 2 MHz, preferably 0.1 to1.5 MHz and most preferably 0.2 to 1 MHz) ultrasound can be applied toproduce the bio-effect mechanisms that enhance the uptake of thereleased or co-administered drug. Exploiting the resonance effects ofthe activated bubbles allows better control of initiation of thesebio-effects at lower acoustic intensities and at lower frequencies thanpossible with other technologies. Coupled with the fact that the largephase shift bubbles are activated and deposited in the tissuemicrovasculature under imaging control (allow spatial targeting of thelarge activated bubbles in tissue), and their prolonged residence time,allows more efficient and controlled implementation of the drug deliverymechanisms. The lower frequency and reduced acoustic powers required byusing the resonance properties of the deposited large phase shiftbubbles has great potential advantage for opening the blood brainbarrier. The lower frequency fields greatly reduce the thermal effectissues currently experienced by other approaches, and the need to removethe skull bone to avoid such issues.

The therapeutic agent, also called “the drug”, to be delivered may beselected from the group of drug molecules, nanoparticles andnanoparticle delivery systems, genes, and radioisotopes. This is eitherdissolved or otherwise incorporated (e.g. dispersed) in the oil phase ofthe second component, or is alternatively administered as a separatecomposition. Examples of the drug classes include, but are not limitedto, genes (for gene therapy), chemotherapeutics, immunotherapeutics(e.g. for cancer therapy or organ transplant therapy), angiogenesisproducing drugs for example to stimulate the growth of new bloodvessels, drugs to pass the blood brain barrier for example to treatcancer or neurological diseases such as Parkinson’s and Alzheimers.

For chemotherapeutics example drugs include, but are not limited to, thedrug classes: Alkylating agents such as Cyclophosphamide,Mechlorethamine, Chlorambucil, Melphalan; Anthracyclines such asDaunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone,Valrubicin; Cytoskeletal disruptors (Taxanes) such as Paclitaxel andDocetaxel; Epothilones; Histone Deacetylase Inhibitors such asVorinostat, Romidepsin; Inhibitors of Topoisomerase I and II such asIrinotecan, Topotecan, Etoposide, Teniposide, Tafluposide, ; Kinaseinhibitors such as Bortezomib, Erlotinib, Gefitinib, Imatinib,Vemurafenib, Vismodegib; Monoclonal antibodies such as Bevacizumab,Cetuximab, Ipilimumab, Ofatumumab, Ocrelizumab, Panitumab, Rituximab;Nucleotide analogs and precursor analogs such as Azacitidine,Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil,Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Tioguanine;Peptide antibiotics such as Bleomycin, Actinomycin; Platinum-basedagents such as Carboplatin, Cisplatin, Oxaliplatin; Retinoids such asTretinoin, Alitretinoin, Bexarotene; Vinca alkaloids and derivativessuch as Vinblastine, Vincristine, Vindesine, Vinorelbine.

As noted in Example 5, in the current invention, hydrophobic drugs witha LogS of less than - 2 are preferred.

In one embodiment of the invention the drug is dissolved in the oilmicrodroplets comprising the diffusable component. In order to dissolvethe drug in sufficient quantity, e.g. greater than 0.5% by mass orpreferably greater than 2% by mass, one or more solvents (co-solvents)may be added to the main oil component. The chemical Reactivity of thesolvents should preferably be inert such as, but not limited to,non-substituted alkanes and ethers, hydrogenated fluoro carbons (hFC)and perfluorinated (pF) alkanes (pFC), pF-cycloalkanes, pF-ethers,hydrogenated fluoro ethers (hFE), pF-oxolanes, pF-furanes, pF-pyranes,diethylsilane. Co-solvents should preferably be restricted to the IHCclasses 2, 3 and 4, Solvents with Low Toxic Potential and Solvents forwhich No Adequate Toxicological Data, such as, but not limited to,acetone, ethanol, ethyl acetate, 2-propanol, 1,1-dimethoxymethane,isopropyl ether, trichloroacetic acid, etc. Further examples ofpotentially preferable solvents include, but are not limited to,dimethylsulfoxide (DMSO), oxetane (trimethyleneoxide),1-chloro-2-fluoroethane, diethyleneglycolmonoethylether,methylenedichloride (dichloromethane), methylenetrichloride(trichloromethane), 3-fluorooxetane, glycofurol, dichloroethylene,1,3-difluoropropane, 2-chloro-1,1-difluoroethane,1-chloro-2,2-difluoroethane, 1,2,2-tetrafluoroethylfluoromethylether,methylisopropylether, 1-propanol, propyleneglycol, 2-propanol,1-pentanol, 1-butanol, 2-butanol, 1,3-butanediol and isobutylalcohol.

To optimize drug-loading capacity, it may be particularly useful to usetwo or more co-solvents simultaneously. Particularly useful co-solventsinclude methylenedichloride, methylenerrichloride and2-chloro-1,1-difluoroethane.

The acoustic signal from the large phase shift bubbles produced uponactivation of the cluster composition at the desired spatial locationcan be measured, typically with a medical ultrasound imaging system, inorder to quantify the amount of drug released by the composition anddelivered to the tissue region. The acoustic information recorded andinterpreted, for example by means of suitable software algorithms andsupport methods using computer processing. Hence, in one embodiment theinvention provides a method of quantification of the amount of drugreleased by analysis of the acoustic signature produced by the largephase shift bubbles liberated by activation of the phase shifttechnology. Hence, the amount of drug delivered is quantified byprocessing of the acoustic signatures of the large, activated bubbles.

In another embodiment, the invention provides a method for delivery ofdrugs as part of a multidrug treatment regime.

In yet another embodiment the invention provides the method including astep of using low MI contrast agent imaging modes (MI < 0.15) to imagethe microbubble component, i.e. the dispersed gas, without activation ofthe clusters to identify the pathology region for treatment. Hence, asthe clusters are not activated at low MI (below the activationthreshold) standard medical ultrasound contrast agent imaging may beperformed, prior to the activation step, for example to identify tumourmicro vascular pathology without activation of the clusters.

In yet another embodiment, the invention provides the use of the deposittracer properties of the activated bubbles and ultrasound imaging toidentify the pathology region for treatment and to quantify perfusion.In addition to the use of low MI contrast agent imaging modes to imagethe microbubble component without activation of the clusters to identifythe pathology region for treatment, the method may include use of thedeposit tracer properties of the activated bubbles to identify thepathology region for treatment and to quantify perfusion.

In yet another embodiment of the invention a therapeutic agent is pre-,and/or co- and/or post administered. The cluster composition isadministered and the activation step is performed. The activation of thecluster composition produces large phase shift bubbles that are trappedat the site of interest temporarily stopping blood flow. Furtherapplication of ultrasound after trapping facilitates bio-mechanisms,such as increasing the permeability of the vasculature, hence increasingthe uptake and/or distribution and hence the efficiency of the pre-,and/or co- and/or post administered drug.

In yet another embodiment of the invention a therapeutic agent is givenboth as loaded into the emulsion microdroplets in the second componentand as a separate composition.

In yet another embodiment of the invention the perfusion of the tissueregion being treated is quantified by processing of the acousticsignatures of the large, activated bubbles.

In yet another embodiment of the invention the acoustic signature of thelarge phase shift bubbles is wholly or partially separated from theacoustic signature of the tissue region by means of processing of thebackscattered signals and used to improve the quantification of the drugdelivered and/or the perfusion of the tissue being treated.

In yet another embodiment of the invention high power ultrasound (HighIntensity Focused Ultrasound, HIFU) [Lukka, H. et al., Clinical Oncology23 (2011) 117-127] is applied to the tissue region containing the large,activated bubbles. The presence of the large phase shift bubblesincreases the local rate of thermal delivery using ultrasoundhyperthermia treatment and/or tissue ablation.

In yet another embodiment of the invention high power ultrasound isapplied to the tissue region containing large phase shift bubbles tolyse cells, for example cancer cells, to invoke a systemic immuneresponse to the cancer tissue.

The cluster composition of the invention can thus be for use as apharmaceutical composition.

In a second aspect, the invention provides a pharmaceutical compositionthat comprises

-   (i) the cluster composition as defined in the first aspect-   (ii) an optional second therapeutic agent, provided either in    mixture with (i), or as a separate composition to (i);-   wherein said pharmaceutical composition comprises at least one    therapeutic agent.

In a first embodiment of the second aspect, the therapeutic agent in thecluster composition of the pharmaceutical composition is absent, butprovided as a separate composition.

In a second embodiment of the second aspect, a first therapeutic agentis present in the cluster composition of the pharmaceutical composition,and a second therapeutic agent is also present and provided as aseparate composition.

In a third aspect, the invention provides an ultrasound contrast agentthat comprises the cluster composition as described in the first aspector the pharmaceutical composition described in the second aspects.

In a fourth aspect, the invention provides a method of delivering atleast one therapeutic agent to the mammalian subject, comprising thesteps of:

-   (i) administering the pharmaceutical composition as defined in the    second aspect to a mammalian subject;-   (ii) optionally imaging the microbubbles of said pharmaceutical    composition using ultrasound imaging to identify the region of    interest for treatment within said subject;-   (iii) activating a phase shift of the diffusible component of the    second component of the cluster composition from step (i) by    ultrasound irradiation of a region of interest within said subject,    such that:    -   (a) the microbubbles of said clusters are enlarged by said        diffusible component of step (iii) to give enlarged bubbles        which are localised at said region of interest due to temporary        blocking of the microcirculation at said region of interest by        said enlarged bubbles; and    -   (b) said activation of step (iii) facilitates extravasation of        the therapeutic agent(s) administered in step (i).-   (iv) optionally, facilitating further extravasation of the    therapeutic agent(s) administered in step (i) by further ultrasound    irradiation.

In this context, in steps ii, iii and iv, ultrasound of any mechanicalindex may be used. However, in step ii a MI of < 0.15 is preferred, andin steps iii and iv a MI of < 0.7 is preferred. In this context, insteps ii, iii and iv, ultrasound of any frequency between 0.05 to 30 MHzmay be used. However, in steps ii and iii a frequency in the range of1-10 MHz is preferred, and in step iv a frequency in the range 0.05-2MHz is preferred.

The pharmaceutical composition is preferably administered to saidmammalian subject parenterally, preferably intravenously. The route ofadministration might also be selected from the intra-arterial,intra-muscular, intra-peritoneal or subcutaneous administration. In afifth aspect, the invention provides a method treatment of the mammaliansubject that comprises the method of delivery as defined in the fourthaspect.

The invention also relates to the use of the pharmaceutical compositionof the invention or the method of delivery of the invention in thetreatment of a mammalian subject.

In a sixth aspect, the invention provides a method of treatment of themammalian subject, which comprises administering the cluster compositionof the invention or the pharmaceutical composition of the invention andapplication of High Intensity Focused Ultrasound (HIFU) to a region ofinterest.

In a seventh aspect, the invention provides use of the clustercomposition of the invention or the pharmaceutical composition of theinvention as an ultrasound contrast agent or medicament.

In an eighth aspect, the invention provides a method of ultrasoundimaging, which comprises imaging a mammalian subject previouslyadministered with the ultrasound contrast agent of the invention

In the second to eighth aspects, the same components and embodiments asdescribed in the first aspect may be used.

EXAMPLES

The following non-limitative Examples serve to illustrate the invention.For simplicity, in all the following examples the 1^(st) component isdesignated C1, the 2^(nd) component is designated C2 and the clustercomposition, i.e. the composition resulting from a combination of the1^(st) and 2^(nd) components, is designated DP (drug product).

Example 1 provides descriptions of analytical methodologies forcharacterisation and quantitation of microbubble/microdroplet clustersin DP, and explains relevant responses and attributes includingconcentration, size and circularity. It also provides details onanalytical methodology for characterisation and quantification ofactivated bubble size and concentration. In addition, data on clusterstability after preparation are presented, as is a comparison ofcharacteristics for pre-mixed vs. co-injected DP. It also detailsengineering steps for controlled manipulations of cluster content andsize in DP.

Example 2 provides results from two in-vivo studies elucidating effectsof cluster characteristics on product efficacy as the ability to depositlarge, activated bubbles in the microcirculation. It further analysethese data and concludes that clusters with a size between 3 to 10 µm,defined by a circularity of less than 0.9, are contributing to theefficacy of the cluster composition. It also compares results on productefficacy with results reported in WO 99/53963 and shows that the currentinvention offers a 10-fold increase in the amount of deposited phaseshift bubbles.

Example 3 provides results from a study demonstrating activated bubblessize and dynamics in-vivo. It confirms the results from the in-vitroanalysis, showing an activated mean bubble size of approx. 20 µm.

Example 4 provides results from an in-vivo study demonstrating thedeposit nature of the activated bubbles by intravital microscopy ofmesentery tissue. It also provides theoretical calculations on thevolume oscillations of the large, activated bubbles upon US irradiationand compares these to volume oscillations of regular contrastmicrobubbles. It concludes that the absolute volume oscillationsprovided by the large, activated bubbles of the current invention isthree orders of magnitude larger than with regular contrastmicrobubbles.

Example 5 provides results from various formulation studies on C1 andC2. It shows that the concept taught by the current invention isfunctional when using commercially available microbubble formulations;Sonazoid, Optison, Sonovue, Micromarker and Polyson as C1, hence provingthat a range of microbubble components can be explored for use in thecurrent invention. Results for cluster compositions made with some ofthese agents demonstrate the clusters down to approx. 1 µm in diametercan be activated and hence contribute to the overall efficacy of thecomposition. Example 5 also investigate a range of diffusible componentsfor use in C2 and shows that spontaneous activation upon mixing of C1and C2 can be avoided by using low water solubility, perfluoratedhydrocarbons and also that use of such compounds increase the ability toform large phase shift bubbles upon US activation. Further, example 5provides data from investigations on drug loading of C2 and the use ofpartially halogenated hydrocarbons as co-solvents to facilitate suchloading.

Example 6 provides results from fluorescence microscopy on activatedbubbles made with C2 loaded with Nile Red fluorescence dye. Itdemonstrate that, after activation, the loaded substance ishomogeneously expressed at the surface of the activated bubbles andhence will be in close contact with the endothelial wall and accessiblefor extravasation.

Example 7 provides results from a US imaging contrast studydemonstrating the deposit nature of the activated bubbles in a murinecancer model, and compares their characteristics with regular HEPS/PFBmicrobubbles (C1). It shows that, upon administration of DP andsubsequent activation, the large phase shift bubbles are deposited inthe tumour microcirculation and remain stationary for several minutes.No change in contrast level is observed 1.5 minutes after activation.Contrary, HEPS/PFB microbubbles show free flowing contrast that washesout rapidly and return completely to base line after less than 1 minute.

Example 8 provides results from investigations of delivery ofco-administered and loaded compounds. In a first study cohort, it isshown that administration of DP with subsequent activation and furtherUS irradiation increases the uptake in muscle tissue by a factor of 2.Using identical US irradiation procedures, no increase in uptake wasobserved after administration of HEPS/PFB microbubbles (C1) only. WithDP, uptake in tumour increased with a factor of 2 upon activation onlyand by a factor of 3.4 after further US irradiation. In a second studycohort, it was shown that administration of DP with subsequentactivation increase the tumour uptake (as increase in luminescenceintensity) of a CW800 IR dye with approx. 30% and that further USirradiation increase the tumour uptake with approx. 60%. In a thirdstudy cohort, administration of DP loaded with a DiR fluorescence dye,subsequent activation and further US irradiation was investigated.Results showed a strong, significant increase in fluorescence intensityin treated tumour tissue, demonstrating release and uptake of thefluorescence dye loaded into the C2 component.

Example 9 provides a description of the manufacture of C1 and C2. Threeconsecutive batches of C1 and C2 passed sterility testing according topharmacopeia (Ph.Eur./USP).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 - Results from Coulter counter analyses on the 1^(st) component(microbubbles, dotted line), the 2^(nd) component (microdroplets,dot-dash line), sum of the 1^(st) and 2^(nd) components (dot-dot-dashline) and the cluster composition (solid line) for three levels ofelectrostatic attraction between the microbubble in the 1^(st) componentand the microdroplets in 2^(nd) component. Y-axis is numberconcentration (a.u.), x-axis is diameter in µm. Low attraction with 1.5%SA (upper plot), medium attraction with 3% SA (middle plot) and highattraction with 5% SA (lower plot). As can be observed the loss in totalnumber of particles in the system (indicated by white arrows) increasesfrom negligible at 1.5% SA to more than 50% at 5% SA. In addition, thelarge end tailing of the size distribution of the cluster composition(indicated by black arrows) increase with increasing electrostaticattraction, demonstrating an increased content ofmicrobubble/microdroplet clusters.

FIG. 2 - Results from Flow Particle Image Analysis on the clustercomposition. Representative selection of micrographs of particlesbetween 5 to 10 µm showing microbubble/microdroplet clusters.

FIG. 3 - Results from microscopy and image analysis on the 1^(st)component (microbubbles, upper plot) and the 2^(nd) component(microdroplets, lower plot). Plot pane numbered 1 shows the sizedistribution of microbubbles/microdroplet, where y-axis is number ofdetections and x-axis is diameter in µm. Plot pane numbered 2 shows thecircularity distribution of microbubbles/microdroplets, where y-axis iscircularity and x-axis is number of detections. Plot pane numbered 3show the size (x-axis) vs. circularity (y-axis) scatter plot where eachdetection is plotted as a single spot in the size/circularity matrix.Greyed area in plot pane 3 designates detections > 3 µm with acircularity < 0.9. Right (large) pane shows a representative selectionof micrographs from individual detections of microbubbles/microdroplets.As can be observed from the upper plot, the microbubbles in the 1^(st)component display a fairly narrow size distribution with a mediandiameter of approx. 2.8 µm as well as a narrow circularity distributionwith a median circularity of approx. 0.98. Less than 1% of thedetections are contained in the diameter > 3 µm and circularity < 0.9sector and all of these are individual microbubbles. As can be observedfrom the lower plot, the microdroplets in the 2^(nd) component display afairly narrow size distribution with a median diameter of approx. 3.0 µmas well as a narrow circularity distribution with a median circularityof approx. 0.96. Less than 1% of the detections are contained in thediameter > 3 µm and circularity < 0.9 sector and all of these areindividual microdroplets.

FIG. 4 - Results from microscopy and image analysis on the clustercomposition, prior to (upper plot) and after (lower plot) US inducedphase shift activation. Plot pane numbered 1 shows the size distributionof detected particles, where y-axis is number of detections and x-axisis diameter in µm. Plot pane numbered 2 shows the circularitydistribution, where y-axis is circularity and x-axis is number ofdetections. Plot pane numbered 3 show the size (x-axis) vs. circularity(y-axis) scatter plot where each detection is plotted as a single spotin the size/circularity matrix. Upper plot, greyed area in plot pane 3designates detections > 3 µm with a circularity < 0.9. Upper plot, right(large) pane shows a representative selection of micrographs fromindividual detections in the diameter > 3 µm and circularity < 0.9sector. Compared to FIG. 3 , the particles in the non-activated clustercomposition display a long end tailing in size and a low end tailing incircularity, observed as a pronounced ridge in the size vs. circularityscatterplot, demonstrating the presence of microbubble/microdropletclusters. Approx. 6% of the detections are contained in the diameter > 3µm and circularity < 0.9 sector. Of these, more than 95% aremicrobubble/microdroplet clusters (i.e. less than 5% individualmicrobubbles or microdroplets). Lower plot, right (large) pane shows arepresentative selection of micrographs from individual detections ofthe large, activated bubbles. As can be observed, upon US irradition theclusters in the cluster composition phase shift to produce a populationof large phase shift bubbles contained between approx. 10 to 100 µm witha median diameter of approx. 20 µm.

FIG. 5 - The relative number size (upper) and circularity (lower)distributions of microbubble/microdroplet clusters isolated from theresults for the cluster composition displayed in FIG. 4 . As can beobserved, the clusters in the cluster composition are ~3 to ~10 µm indiameter and are characterised by a circularity of < 0.9.

FIG. 6 - Responses from Sonometry analysis. Left plot: volume fraction(y-axis) in the Sonometer measuring cell vs. time (x-axis) afteractivation. Right plot: volume weighted (A) and number weigther (B) meandiameter (y-axis) of activated bubbles vs. time (x-axis) afteractivation.

FIG. 7 - Stability of the cluster composition. Concentration of clustersbetween 5 to 10 µm from FPIA analysis (open circles, left axis) andactivated bubble volume per microdroplet volume from Sonometry analysis(filled circles, right axis) vs. time after preparation.

FIG. 8 - Volume weighted median diameter (y-axis, µm) of activatedbubbles from Sonometry analysis vs. the Reactivity (x-axis, %) of thecluster composition from Coulter analysis.

FIG. 9 - Efficacy of the cluster composition vs. Reactivity. Y-axisshows linear enhancement in the US signal from dog myocardium (GreyScale units) upon i.v. administration of the cluster composition andactivation in the left ventricle. X-axis shows the Reactivity (%) of thecluster composition from Coulter analysis. These results demonstrateoptimal Reactivity between 30 to 60%.

FIG. 10 - Cluster size in the cluster composition vs. Reactivity. Y-axisshows content of clusters in % average value observed for size classes;< 5 µm (dotted line), 5 to 10 µm (solid line), 10 to 20 µm (dash-dotline) and 20 to 40 µm (dash-dot-dot line). X-axis shows the Reactivity(%) of the cluster composition from Coulter analysis. These resultsdemonstrate the shift towards larger clusters with increased Reactivityand the depletion of small and medium sized clusters at high Reactivity.

FIG. 11 - Efficacy of the cluster composition vs. cluster concentrationand activated bubble volume. Left hand figure; Y-axis shows linearenhancement in the US signal from dog myocardium (Grey Scale units) uponi.v. administration of the cluster composition and activation in theleft ventricle. X-axis shows concentration (millions/mL) of clustersbetween 5 to 10 µm in the administered cluster composition from FPIAanalysis. Right hand figure; Y-axis shows linear enhancement in the USsignal from dog myocardium (Grey Scale units) upon i.v. administrationof the cluster composition and activation in the left ventricle. X-axisshows activated bubble volume (µL/mL) in the administered clustercomposition from Sonometry analysis. Greyed area in both figuresindicate range of myocardial enhancement reported in WO 99/53963. Theseresults demonstrate that the in-vitro parameters investigated are goodpredictors for the efficacy of the composition, and that the efficacy ofa cluster composition as taught by the present invention offers a10-fold increase in efficacy vs. what is taught in WO 99/53963.

FIG. 12 - Results from multivariate, principal component analysis (PCA)of the contribution of clusters in various size classes to the linearenhancement in the US signal from dog myocardium (Grey Scale units) uponi.v. administration of the cluster composition and activation in theleft ventricle. The PCA was performed on data for the 30 samplesdetailed in Tables 7 and 8. Left hand plot; Y-axis shows the calculatedcorrelation coefficient, i.e. the contribution to myocardial enhancementfor cluster size classes (X-variables) < 5 µm, 5 to 10 µm, 10 to 20 µmand 20 to 40 µm. Right hand plot; Y-axis shows the calculated myocardialenhancement (GS units) using the model from the left hand plot andX-axis shows measured myocardial enhancement (GS units) for each sample(R=0.85). These results demonstrate that small and medium sized clusters(< 10 µm) contribute significantly to the efficacy of the clustercomposition whereas larger clusters (> 10 µm) do not.

FIG. 13 - The relative number size (upper) and circularity (lower)distributions of microbubble/ microdroplet clusters isolated from theresults for the cluster composition with a 46% Reactivity (sample number3 from Table number 4). As can be observed, the clusters in the clustercomposition are ~3 to ~10 µm in diameter and are characterised by acircularity of < 0.9.

FIG. 14 - Volume fraction (y-axis, ppm) of activated bubbles in arterialblood vs. time (x-axis, seconds) after i.v. administration of a clustercomposition and activation in the heart chamber.

FIG. 15 - Top left micrograph shows an image of rat mesentery 17 secondspost-injection and activation of the cluster composition in themesentery with a phase shift bubble temporarily lodged in themicrovasculature blocking blood flow. The area indicated by the dashedrectangular box is shown schematically in the illustration (bottomleft). The outline of the phase shift bubble has been zoomed by a factorof 5. The outline of the phase shift bubble is labelled A with a 20micron scale bar labelled C. A 3 µm HEPS/PFB microbubble, labelled B andshown to scale, is clearly small enough not to block the vessel in thesame manner as the activated phase shift bubble. (Top right) and (bottomright) show the same region of mesentery at 5 minutes and 19 seconds and5 minutes and 45 seconds post injection respectively. The phase shiftbubble shrinks and moves intermittently down the vascular tree beforedethatching completely, being washed out by the reestablished bloodflow.

FIG. 16 - A 30 micron diameter phase shift bubble labelled A, and a 3micron diameter HEPS/PFB microbubble labelled B, to scale with a 10micron scale bar. The minimum and maximum diameters of the simulatedresponses to US insonation are depicted by the smaller and largerdiameter dashed lines also drawn to scale. There is an approx. 3 ordersof magnitude increase in the absolute change in volume due to theinsonation of the phase shift bubble compared to the HEPS/PFBmicrobubble demonstrating a fundamental difference in the mechanicaleffects on surrounding tissue between these two bubble types.

FIG. 17 - Effect of oil phase water solubility on spontaneous and USactivated bubble growth. Left hand plot; Y-axis show score frommicroscopy examination for amount of large phase shift bubbles formedspontaneously upon mixing the 1^(st) component and the 2^(nd) component(i.e. in the cluster composition) (0 = no or very low number ofbubbles > 15 µm observed, 1 = medium number of bubbles > 15 µm observedand 2 = high number of bubbles > 15 µm observed). X-axis shows molarwater solubility of the oil phase in the 2^(nd) component. Right handplot; ; Y-axis show score from microscopy examination for amount oflarge bubbles formed upon US activation of the cluster composition (0 =no or very low number of bubbles > 15 µm observed, 1 = medium number ofbubbles > 15 µm observed and 2 = high number of bubbles > 15 µmobserved). X-axis shows molar water solubility of the oil phase in the2^(nd) component. These results demonstrate that the level ofspontaneous activation increases, and that the level of US inducedactivation decreases, with increasing water solubility of the oil phasein the 2^(nd) component.

FIG. 18 - Micrographs of 2^(nd) components loaded with DiR dye, Nile Reddye and Paclitaxel showing stable emulsions with no sign ofprecipitation of load molecules and a microdroplet diameter in the 1 to5 µm size range.

FIG. 19 - Micrograph from fluorescence microscopy on an intersection ofactivated phase shift bubbles from a cluster composition where themicrodroplets in the 2^(nd) component was loaded with 5 mg/mL Nile Reddye. As can be observed, after activation the molecular dye loaded intothe microdroplets is expressed at the surface of the activated bubbleand will hence be in close contact with the endothelial wall andaccessible for extravasation.

FIG. 20 - Left hand image shows a typical ultrasound image of a PC-3subcutaneous tumour in the hind limb of a mouse. The dashed white lineindicates the location of the tumour tissue. The interior of the tumouris typicaly hypoechogenic when compared to surrounding tissue such asskin and muscle. The right hand image shows a typical ultrasound imageof the same PC-3 tumour as shown on the left image, after i.v. injectionand activation of the cluster composition. The additional contrastechoes which are clearly depicted in the tumour interior are depositedin the tissue and remain stationary in the tumour tissue for severalminutes.

FIG. 21 - Typical time intensity curves (TIC) of contrast enhancement ina PC-3 tumour after administration and activation of the clustercomposition (A) and after an equivalent dose of HEPS/PFB microbubbleonly (B), measured in the same tumour. The linearised backscatterintensity is averaged in a region of interest covering the tumourcentre. The y-axis is the value of the averaged linearised backscatter,and the x-axis is the video frame number in the video sequence. Thevideo was acquired at a rate of 10 frames per second. As can beobserved, with the cluster composition the scattering intensity peaks ata high level after approx. 20 s and remains stable over the investigatedtime span. Contrary, administered with the HEPS/PFB microbubbles only,the scattering intensity peaks at a lower level than with the phaseshift bubbles and depletes back to baseline after approx. 1 minute.

FIG. 22 - Typical epifluorescence images for co-administration of theLiCor CW800 EPR agent with the cluster composition. An animal from group1 (left image) received no US irradiation whereas an animal from group 3(right image) received US activation and subsequent low frequency USirradiation. The arrows indicate the location of the tumours which areboth approximately the same size and location on each animal. The imageswere taken with the same scanner settings and are presented with thesame fluorescence intensity linear grey scale for direct comparison.There is a clear increase in fluorescence intensity from the tumourreceiving ultrasound activation and subsequent US irradiation comparedto the tumour which received no ultrasound irradiation, demonstrating asignificantly increased uptake of the CW800 dye when treated with thecluster composition.

FIG. 23 - Ratio of tumour fluorescence intensity to untreated controlleg intensity from 1 minute to 9 hours post treatment. The y-axis is theratio of tumour intensity to untreated control leg intensity. The x-axisis time in minutes. There is statistically significant increased initialuptake in group 2 (squares; activation only) compared to group 1(diamonds; no activation, no subsequent US irradiation), andstatistically significant increased initial uptake and uptake rate ingroup 3 (circles; activation and subsequent US irradiation), compared togroups 1 and 2.

FIG. 24 - Ratio of the average intensity in the tumour region to theaverage intensity in the untreated leg was integrated from 1 minute to 1hour post treatment. Groups A, B and C are “no activation, no subsequentUS irradiation”, “activation only” and “activation and subsequent USirradiation, respectively. The observed increase in uptake isstatistically significant between groups A and B and between groups Band C.

FIG. 25 - Typical post-treatment epifluorescence images with a clustercomposition where the microdroplets in the 2^(nd) component were loadedwith 10 mg/mL DiR dye. Upper image (A) is from an animal from group 1receiving no activation or subsequent US irradiation to the left tumourbearing leg. Lower image (B) is from an animal from group 2 where thecluster composition was activated followed by subsequent US irradiationto the left tumour bearing leg. The location of the tumour is indicatedby the arrow. The observed differences in fluorescence intensity clearlydemonstrate release and tissue uptake of the loaded molecular dye uponactivation and subsequent US irradiation, as shown by the statisticalanalysis given in Table 22.

EXAMPLE 1 (E1) - ANALYTICAL TOOLS AND BASIC CHARACTERISTICS OF THEINVENTION E1-1 Introduction

The microbubble/microdroplet clusters formed upon combining C1 and C2,i.e. present in DP, are crucial to the critical quality attributes ofthe composition, i.e. its functionality for delivery of drugs. Hence,analytical methodology to characterize and control the clusters formedwith regards to concentration and size, is an imperative tool to assessthe current invention as well as for medicinal Quality Control (QC). Wehave identified three different analytical tools that can be applied forthis purpose; Coulter counting, Flow Particle Image Analysis (FPIA) andMicroscopy/Image analysis. In the following text, these three analyticalmethodologies and suitable responses are briefly explained, some basiccharacteristics of C1, C2 and DP are exemplified, as are some aspectsfor controlled engineering of these characteristics.

In addition to these techniques, applied for characterisation of theclusters in the cluster composition, analytical methodology has beendeveloped to study the activation of the clusters in vitro, i.e. thegeneration of large, activated bubbles upon US irradiation. Thismeteorology; “Sonometry” is detailed in E1-6. Primary report responsesfrom the Sonometry analysis are number and volume of activated bubblesand their size distribution, both vs. time after activation. Activationresponses may also be explored by Microscopy/Image analysis as detailedin E1-5.

E1-2 Components and Compositions Investigated

The 1^(st) component (C1) in all the compositions investigated in allexamples with exception of E5-2, consisted of per-fluorobutane (PFB)microbubbles stabilised by a hydrogenated egg phosphatidyl serine-sodium(HEPS-Na) membrane and embedded in lyophilized sucrose. HEPS-Na carriesa negatively charged head group with an ensuing negative surface chargeof the microbubbles. Each vial of C1 contains approximately 16 pL or2·10⁹ microbubbles, with a mean diameter of approximately 2.0 µm.

The 2^(nd) component (C2) in the all the compositions investigated inthis example consisted of perfluoromethyl-cyclopentane (pFMCP)microdroplets stabilised by a1,2Distrearoyl-sn-glycerol-3-phosphocholine (DSPC) membrane with 3%mol/mol stearlyamine (SA) added to provide a positive surface charge.The microdroplets in the C2 were dispersed in 5 mM TRIS buffer. Thestandard formulation of C2 investigated in these studies containsapproximately 4 µL or 0.8·10⁹ microdroplets per mL, with a mean diameterof approximately 1.8 µm.

In some cases, to elucidate effects on cluster characteristics, avariety of formulation variables such as SA content, microdroplet size,microdroplet concentration, TRIS concentration and pH was varied in acontrolled manner. In case such samples have been used, these aspectsare detailed in the text.

The cluster composition (DP) was prepared aseptically by reconstitutinga vial of C1 with 2 mL of C2 followed by 30 s manual homogenisation. 2mL was withdrawn from a vial of C2 using a sterile, single use syringeand needle. The content of the syringe was added through the stopper ofa vial of C1 and the resulting DP was homogenised.

C1 and C2 manufactured as detailed in Example 9.

In some cases, in order to compare effects of the cluster composition ofthe current invention to regular contrast microbubbles, C1 was preparedwith pure water instead of C2 to produce an aqueous dispersion ofHEPS/PFB microbubbles.

E1-3 Coulter Counting

Coulter counting is one of the most widely used analytical technique forquantification and size characterization of particulate substanceslarger than 1 µm and has been shown suitable for QC of medicinal drugproducts [Sontum, PC. and Christiansen, C., J. Pharm. Biomed. Anal. Vol.12, No.10, 1233-1241 (1994)]. In brief, a small aliquot of the analyte(e.g. C1, C2 or DP) was diluted/dispersed in a particle free aqueouselectrolyte (typically phosphate buffered saline, PBS) and homogenizedby continuous stirring. A portion of the diluted sample was then drawnthrough an aperture in the instrument, over which the resistivity iscontinuously measured. Each particle that is drawn through the aperturewill cause the resistivity to change proportionally to the volume of theparticle. During the course of the analysis, the instrument draws aknown volume of electrolyte through the aperture, measures and countseach resistivity pulse, and presents the results as number concentrationof particles measured vs. size. For the reported analyses a CoulterMultisizer III or IV (Beckman Coulter Ltd.) set up with a 50 µm aperture(measuring range 1 to 30 µm) was utilized. A suitable sample volume wasdiluted in Isoton II (PBS electrolyte, Beckman Coulter Ltd.) andhomogenized by continues stirring throughout the analysis.

Coulter counting is suitable for quantification of microbubble andmicrodroplet concentration and size distribution in C1 and C2, and forcharacterization of particles in DP. As two or moremicrobubbles/microdroplets in a cluster are counted as a singleparticle, the formation of clusters upon combining the two componentswill lead to 1) a reduction in the total number of particles in thesystem and 2) a shift towards larger sizes. These effects areexemplified in FIG. 1 showing the concentration and size distribution ofC1 and C2 as individual components, the sum C1 and C2 (i.e. the combinedcomposition had there been no formation of clusters upon mixing) and ofDP. Plots are showing results using a C2 formulation with 1.5%, 3.5% and5.5% SA, a positively charged surfactant, in the stabilizing membrane.The amount of SA affects the surface charge (zeta potential) of themicrodroplets and the strength of the attractive electrostatic forcedbetween the microdroplets in C2 and the microbubbles in C1, and hencethe ability to form clusters upon mixing. The zeta potential of themicrodroplets in these three samples was measured to +22 mV, +35 mV and+43 mV for the 1.5%, 3.5% and 5.5% SA formulations, respectively. Allsamples were made with the same C1 formulation. The zeta potential ofthe microbubbles in C1 was measured to -57 mV. As shown in FIG. 1 , alarger difference in surface charge between the two components (i.e.larger attractive electrostatic forces) leads to the formation of moreand larger clusters. With the 1.5% SA formulation, there was aninsignificant change in concentration and size in DP from thetheoretical sum of C1 and C2, hence no evidence of cluster formation wasobserved. On the other hand the 3.5% SA formulation shows a slight, butsignificant decrease in concentration and increase in large end tailingand the 5.5% SA formulation shows evidence of significant clustering,with a marked decrease in number concentration and a clear shift towardsa stronger large end tailing. The attractive force between themicrobubbles and microdroplets hence needs to be over a certainthreshold in order for stable clusters to be formed upon combination ofC1 and C2.

A particularly useful response from these measurements is the Reactivity(R) of the cluster composition defined as;

R =(C_(C1) + C_(C2)- C_(DP)) ⋅ 100/(C_(C1) + C_(C2))

Where C_(C1),C_(C2) and C_(DP) are the number concentration observed inC1, C2 and DP, respectively (in C1, then after reconstitution in 2 mL ofpure water). This Reactivity is hence a measure of how many of theindividual microbubbles and microdroplets in C1 and C2 that arecontained in cluster form in the DP. The Reactivity is also correlatedto how large these clusters are (i.e. how many individual microbubblesand microdroplets comprises a single cluster), see E2-5 for furtherdetails. E.g. if there are no clustering then C_(DP) = C_(C1) + C_(C2)and R = 0% and if all the microbubbles and microdroplets in a clustercomposition form a single, large cluster then C_(DP) ~ 0 and R ~ 100%.From Coulter analysis of C1 (after reconstitution in 2 mL of water), C2and DP, R can easily be calculated.

Even though the Coulter analysis is suitable for characterization of thetotal particle concentration and size distribution in DP, it does not,per se, discriminate between microbubbles, microdroplets or clusters;all entities are counted and sized as “a particle”. In order todifferentiate and characterize the clusters specifically, microscopytechniques are necessary.

E1-4 Flow Particle Image Analysis

Flow Particle Image Analysis (FPIA) is a fully automated microscopy andimage analysis technique [Sontum, PC. and Martinsen, E., Abstracts ofEur. Conf. Drug Deliv. Pharm. Tech., Seville (Spain), pp 47, # 25(2004)]. In brief, a small aliquot of the analyte (e.g. C1, C2 or DP)was diluted/dispersed in a particle free aqueous diluent (water or PBS)and homogenized by continuous stirring. A known portion of the dilutedsample was then drawn through a measuring cell in the instrument where afixed set of micrographs are taken by a CCD camera with a stroboscopiclight source. The particles in each frame are automatically isolated andanalyzed by the image analysis software, and a variety of morphologicalparameters are calculated for each particle. In addition, the particleconcentration was reported. Of particular interest to the currentinvention are parameters that discriminate between free microbubbles ormicrodroplets and clusters of the same. For this purpose the particlesize, described as circular equivalent diameter, and their circularityhas been used as standard responses. Circular equivalent diameter isdefined as the diameter of a circle with an equivalent area as theparticle detected. The term “circularity” (C) has its conventionalmeaning in the field of image analysis and is defined on page 12.

In addition to numerical responses, the instrument provides arepresentative selection of micrographs for different size classes; < 5µm, 5 to 10 µm, 10 to 20 µm and 20 to 40 µm. For the reported analyses aSysmex 2100 instrument (Malvern Instruments Ltd.) set up with a HighPower Field (20X) and measuring range 0.7 to 40 µm was utilized. Asuitable sample volume was diluted in water and homogenized by continuesstirring throughout the analysis.

A representative selection of micrographs of individual clusters in thesize class between 5 and 10 µm, from analysis of a DP sample made withC2 containing 3.5% stearlyamine is shown in FIG. 2 . As can be observed,in this size class all detections but one of 117 (i.e. < 1%) aremicrobubble/microdroplet clusters. Table 1 states the numberconcentration of clusters observed in different size classes for thesamples with variable amount of stearlyamine (1.5 to 5.5 %) visualizedin FIG. 1 . As can be noted, corroborating the results in FIG. 1 , thecluster concentration at 1.5% stearlyamine was negligible, at 3.5% asignificant number of small (i.e. < 5 µm) and medium (i.e. 5-10 µm)sized clusters are observed and at 5.5% a decrease in small, and anincrease in the concentration of medium and large (i.e. > 10 µm)clusters are observed.

TABLE 1 Reactivity (R) and concentration (millions/mL) ofmicrobubble/microdroplet clusters in various size classes in the clustercomposition at three levels of electrostatic attraction from variableamounts of stearlyamine (SA%) in the stabilizing membrane of themicrodroplets S A% R (%) <5 µm 5-10 µm 10-20 µm 20-40 µm 1.5 4 6.5 0.00.0 0.0 3.5 21 112.6 3.8 0.1 0.0 5.5 50 84.7 14.0 2.3 0.1

E1- 5 Microscopy / Image Analysis

As an alternative to the FPIA analysis a more manual microscopytechnique coupled with an image analysis software may be employed. Forthis purpose, a Malvern Morphology G3 system (Malvern Instruments Ltd.)with a 20X objective and a measuring range of 1.8 to 100 µm wasutilized. In some cases a 50X objective with a measuring range of 0.5 to40 µm was utilized. In brief, a small aliquot of the analyte (e.g. C1,C2 or DP) was diluted/dispersed in a particle free aqueous diluent (e.g.water or PBS) and homogenized. The diluted sample was then introducedinto a microscopy channel slide (IBIDI µ-slide, IBIDI GmBh), with aknown channel height of 400 µm and placed under the microscope. Theinstrument automatically scans a preset area of the slide and a fixedset of micrographs are taken by a CCD camera. The particles in eachframe are automatically isolated and analyzed by the image analysissoftware, and a variety of morphological parameters are calculated foreach particle. The total number of particles are reported and from theknown scan area and known channel height, the concentration of particlesin the analyte can be calculated. As for the FPIA analysis, the circularequivalent diameter and particle circularity was reported. Micrographsof all particles detected can be displayed and evaluated by manual,visual inspection. Hence all clusters can be isolated from e.g. freemicrobubbles and a full cluster size and circularity distribution can beconstructed for the clusters in each sample.

This methodology can also be used to characterize the activated bubblepopulation, i.e. the cluster composition after ultrasound activation.For this purpose the microscopy slide was immersed in 37° C. water andinsonated for 10 s with an ATL 3-2 transducer (center frequency of 2.25MHz) at a nominal MI of 0.8. Immediately after activation, the slide wasplaced under the microscope and the analysis was repeated.

Typical examples of output from this analysis are shown in FIG. 3 for C1and C2 and FIG. 4 for DP pre- and post- activation. As can be observedfrom the Circularity vs Diameter scatter plots, C1 and C2 display anarrow size distribution with essentially spherical particles whereasnon-activated DP contain a ridge of material with lower Circularitycaused by the presence of microbubble/microdroplet clusters. A visualinspection of all micrographs show that no microbubble/microbubble ormicrodroplet/microdroplet agglomerates are observed in the neat C1 andC2 samples; all detected particles consist of single spherical entities.The results for the DP sample after US irradiation clearly shows thatthe clusters have been activated and have phase shifted to large (> 10µm) gas bubbles. No large gas bubbles are observed after equivalentinsonation of neat C1 or C2 samples alone. FIG. 5 shows results for theentire cluster population, isolated from the DP sample in FIG. 4 . Ascan be observed the clusters are contained between ~3 to ~10 µm and arecharacterized by a circularity < 0.9.

E1-6 Sonometry

In order to study and demonstrate the characteristics of large phaseshift bubbles produced after activation of the microbubble/microdropletclusters present in the composition, an analytical methodology thatallows for determination of activated bubble concentration, size anddynamics after activation in a relevant in-vitro system has beendeveloped. The text below describes in detail a method for sizing theactivated bubbles in vitro, which produces a measurement of theactivated bubble concentration and size distribution from 4 to 80microns in diameter over time. Measurements are performed every 15seconds for a period that cover the time of activated bubble growth anddissolution.

An acoustic transmission technique was used to measure the sizedistribution dynamics of the activated, large bubble populationin-vitro. The acoustic technique requires the measurement of attenuationover a range of frequencies, which are an order of magnitude lower(around 0.2 MHz) than those used for conventional imaging (1-10 MHz).The subsequent conversion to activated bubble size information is basedon bubble resonance theory and the solution of the resulting Fredholmintegral equation of the first kind, using standard techniques. Theassociated velocity dispersion data are used to provide a quantitativequality metric with which to assess the performance of the inversionprocedure. The technique is based on methods described in the sonarliterature to size bubble populations in the upper ocean, withinessential modifications to suit the problem at hand.

In order to obtain information regarding activated bubble size, theprinciple acoustic properties are measured as a function of frequency.This data is then inverted to provide size information. The inversionrequires an accurate model of the interaction of activated bubbles withthe incident sound field. A number of models for the propagation ofnonlinear pressure waves in bubbly liquids are available in theliterature. Here we restrict measurements to the propagation of lowamplitude acoustic waves, which effectively places measurements in thelinear region, hence a linear model is employed. We will also restrictconsideration and measurements to bubble densities for which the Foldyapproximation [Phys. Rev. B, vol. 67, pp. 107-119, 1945] is applicable.Relevant theory is presented in [J. Acoust. Soc. Am 85, pp. 732-746,1989].

A low frequency (Panametrics Videoscan SN:267202 part #V1012, 0.25 MHzcentre frequency) broadband pulse is directed through a sample cell,reflected from a steel plate (approximately 25 cm from the low frequencytransducer), propagates back through the sample and is received by thesame transducer. Thus the pulse passes through the sample cell twice.The internal dimensions of the sample cell are: width 7.4 cm, thickness3.1 cm, height 10.3 cm, giving a total volume capacity of 236.28 cm3.The cell is closed and contains no headspace so that it may be kept at acontrolled gas saturation. The temperature to perform the measurementsis chosen to be 37° C. to mimic body temperature. The gas saturation inthe blood in-vivo is approximately 98 kPa in arterial blood and 90 kPain venous blood. Coupled with systemic overpressure (~ 100 mmHg) thisprovides a gas saturation environment of approximately 85% in-vivo. Thegas saturation of the sample cell was controlled at 85% to mimic thein-vivo environment. Gentle stirring is incorporated to ensure adequatemixing. Mylar membranes are used to provide acoustically transparentwindows. The low frequency source does not activate the clusters.Activation is provided by the high frequency transducer. The bandwidthof the low frequency pulse is able to cover a activated bubble sizerange from 4 to 80 µm in diameter.

The inversion procedure is ill-posed in the sense of Hadamard andtherefore requires optimisation of the data signal-to-noise ratio. Henceit is appropriate to average as much as is practically possible. 200consecutive rf A-line signals are recorded at 10 MHz sampling frequencyto a nominal 8 bits and comprise one measurement data set. The pulserepetition frequency of the transmission transducer is set to 200 Hz,and thus one second is required for data capture. Data sets are recordedonce every 15 seconds and downloaded to a PC for subsequent numericalinversion. 45 such measurement data sets comprise one run, spanning 11minutes in total.

Inverting the measured primary acoustic properties to yield activatedbubble concentration and size distribution information is based on asimple finite element solution as proposed by Commander and McDonald [JAcoust. Soc. Am. 89 pp. 592-597, 1991]. Details of the inversionalgorithm used can be found in [“Solving least squares problems”,Prentice Hall, Chapter 23, p. 161, 1974].

From the acoustic measurements, acoustic attenuation and velocity as afunction of frequency may both be calculated. The velocity data may beregarded as independent to the attenuation data. Only attenuation datais used to calculate the activated bubble size distribution, thevelocity data can be used as the basis of an independent check of theestimated activated bubble size distribution. The velocity of a bubblyliquid is highly dispersive around the resonance frequency. Thisphenomenon may be used to derive a ‘quality’ metric in order toquantitatively infer the accuracy or confidence of the estimatedactivated bubble size distribution, after [IEEE J. of OceanicEngineering, vol. 23, no. 3, 1998].

Primary reports from the meteorology detailed above are activated bubblenumber and volume concentration, and number and volume weighted bubblediameter, both vs. time after activation.

Applying the Sonometry analysis, a sample of the cluster compositiondetailed in E1-2 was analysed. FIG. 6 (left hand) shows the activatedbubble volume concentration (% v/v) in the measuring cell (y-axis) andFIG. 6 (right hand) show the number and volume weighted mean diameter(y-axis), both as a function of time after activation (x-axis). Thequality metric confirmed that the presented size distributions arerobust. As can be observed from FIG. 6 the results generated confirmthat the clusters in the composition are activated within the desired MIrange and produces bubble growth within the desired size range anddynamics in a relevant in-vitro measuring system.

In following examples, primary responses evaluated from this analysisare peak activated bubble volume per microdroplet volume or per volumeof DP, and volume weighted mean diameter at peak activated volume.

E1-7 Stability of Clusters in the Cluster Composition

The clusters in the DP are formed and kept by the electrostaticattraction between the microbubbles and the microdroplets. These forcesare finite and the clusters may break up after formation through variousroutes/influences such as mechanical stress or thermal (Brownian)motion.

For precise and accurate characterization, it is important that theclusters remain stable during the time of analysis. This stability hasbeen investigated with all the methodologies described above. Toevaluate stability, 3 to 5 analyses where repeated on a single DP samplecovering a timespan of > 5 minutes. No significant change in neitherconcentration nor size has been observed cross these replicates, provingthat the microbubbles, microdroplets and clusters are stable for > 5minutes under the analytical conditions stated, i.e. after dilution inPBS or water and under continuous homogenization (stirring).

For use as a medicinal drug product it is imperative that the vitalcharacteristics of the product are kept for a time that enables use. Thestability of DP after preparation has been studies with varioustechniques including FPIA and Sonometry. FIG. 7 shows the clusterconcentration between 5 to 10 µm from the FPIA analysis and the peakactivated volume per microdroplet volume from the Sonometry analysis intwo DP samples, stored at ambient room temperature and pressure, versustime after preparation. No evidence of spontaneous activation wasobserved during the FPIA analysis. As can be observed from FIG. 7 , anegligible change in cluster content and activated bubble volume isobserved over a period of 1h after preparation of DP.

E1-9 Formulation Aspects

A number of different formulation aspects can be explored forcontrolling the cluster content and size in the DP and for targetingoptimal properties. Parameters that can be used to engineer clustercontent and size distribution include, but are not limited to; thedifference in surface charge between the microbubbles and themicrodroplets (e.g. SA% as shown in E1-3): the microdroplet size of C2:the pH: the concentration of TRIS in C2: and the concentration ofmicrobubbles and microdroplets. In addition, chemical degradation of thecomponents, e.g. during prolonged storage at high temperatures, mayinfluence the ability of C1 and C2 to form clusters during preparationof the DP. A brief description of these aspects are given in thefollowing.

Microdroplet size - Samples of C2 with variable microdroplet size wasmade from a single batch of raw emulsion by centrifugation and controlremoval of supernatant and/or sediment after different centrifugationtimes. After size adjustment, the concentration of all samples wasadjusted to the same volume concentration of microdroplets (approx. 4 µlmicrodroplets/mL). C2 samples with microdroplet size as volume mediandiameters of 1.8 µm, 2.4 µm and 3.1 µm were prepare and used forpreparation of DP with vials from a single batch of C1 and theReactivity was measured by Coulter counting. The Reactivity was found toincrease with decreasing microdroplet size, from 27% at 3.1 µm, to 49%at 2.4 µm and 78% at 1.8 µm. Decreasing microdroplet size hence increaseformation of clusters upon mixing of C1 and C2.

pH - two vials of DP were prepared to a pH of 6.6 and two vials wereprepared to a pH of 6.1. The resulting Reactivity, as measured byCoulter counting, was 30-32% and 51-52% for the pH 6.6. and pH 6.1samples, respectively. A decrease in pH hence increase formation ofclusters.

TRIS concentration - for three samples of C2 from the same batch, theconcentration of TRIS was varied from 1 mM to 10 mM. Using a singlebatch of C1, each sample was used to prepare DP and the concentration ofclusters between 5 to 10 µm was measured by FPIA analysis on allsamples. The formation of clusters was found to decrease with increasingTRIS concentration, with a lowering of cluster concentration from 6.7 to3.7 millions/mL, going from 1 to 10 mM TRIS in C2.

Microdroplet concentration - The formation of clusters upon combining C1and C2 is also a function of the concentration of microbubbles andmicrodroplets in the two components, i.e. the ratio of microbubbles tomicrodroplets. From an intuitive perspective, it seems likely that in asystem where the total surface charge presented by the two componentsbalance completely, the result would be that all microbubbles and allmicrodroplets would form a few, very large clusters (i.e. resulting in atotal collapse of the system). We have found that in order to generate acontrolled and targeted clustering where most all of the microdropletsare contained in cluster form, and were the clusters formed are of anacceptable size, the total charge presented by the microbubbles shouldbe in excess of the total charge presented by the microdroplets.However, the microdroplet/microbubble ratio must also be above a certainthreshold in order to form a significant amount of clusters. Results inTable 2 shows the effect of microdroplet concentration in C2, when usedto prepare DP with a fixed concentration of microbubbles in C1 (8 µlmicrodroplets/mL). As can be noted, we find a strong increase inclustering in terms of Reactivity, and a strong increase in mean clusterdiameter, with increasing microdroplet concentration added to a fixedamount of microbubbles.

TABLE 2 Reactivity (R) from Coulter analysis and mean cluster diameterand from microscopy/image analysis in cluster compositions made with C2with variable microdroplet concentration (C) C (µl/ML) R (%) Meancluster Diameter (µm) 1.0 8 4.9 1.5 26 NA 3.0 46 5.8 6.0 74 NA 9.0 938.5

Thermal degradation - the SA component in the phospholipid membranestabilizing the microdroplets in C2 is prone to thermal degradation uponprolonged storage at elevated temperatures, losing its positive chargein the degradation process. The ability to form microbubble/microdroplet clusters when combined with C1 is hence reduced. Twosamples of C1 was place under controlled storage conditions at 4 and 30°C. for three months and used for preparation of DP on which the contentof clusters between 5 to 10 µm was measured by FPIA analysis. Thecluster contents observed was 29.6 and 0.2 millions/mL for the 4 and 30°C. stored samples, respectively.

E1-10 Size of Activated Bubbles

The size of the activated bubbles may be of importance to the biologicalattributes of the administered composition, e.g. safety and efficacyaspects. Whereas naturally depended upon the size of the microdropletsin C2, the activated bubble size is also strongly related to the clustersize. FIG. 8 shows the covariance between Reactivity by Coulter analysisand activated bubble diameter by Sonometry, measured on the samplesdetailed in Table 4, E2-3. As can be noted the activated bubble diameterincreases from approx. 20 µm at low Reactivity (i.e. from smallclusters) to approx. 50 µm at high Reactivity (i.e. from largeclusters).

EXAMPLE 2 (E2) - IN-VIVO STUDIES ON CLUSTER ATTRIBUTES VS PRODUCTEFFICACY E2-1 Introduction

Having shown in E1 how to measure important characteristics of thecurrent invention; i.e. for the clusters in the cluster composition, andalso how to manipulate and control these, the current example explorewhich cluster characteristics should be targeted for optimal in-vivoefficacy. In order to reach this objective, two extensive in-vivostudies (Study A and Study B) were performed where the US contrastenhancement obtained after administration of a number of DP samples withdifferent characteristics, was measured in an open chest dog myocardiummodel. The myocardial enhancement of the US signal was observed afteri.v. injection and activation of the composition in the left ventricle.After activation the large phase shift bubbles are trapped in themyocardium capillary network and the US contrast enhancement is a directmeasure of the amount of activated bubbles deposited, and hence ameasure of the efficacy of the administered sample.

E2-2 Components and Compositions Investigated

The 1^(st) component (C1) in the compositions investigated in thisexample is described in E1-2.

The 2^(nd) component (C2) in the all the compositions investigated inthis example consisted of perfluoromethyl-cyclopentane (pFMCP)microdroplets stabilised by a1,2Distrearoyl-sn-glycerol-3-phosphocholine (DSPC) membrane withstearlyamine (SA) added to provide a positive surface charge. Themicrodroplets in the C2 were dispersed TRIS buffer.

In order to obtain a significant variance in the cluster characteristicsof the cluster composition (DP) formulation variables such as SAcontent, microdroplet size, microdroplet concentration, TRISconcentration and pH was varied in a controlled manner, as described inE1.

In Study A the microdroplet size, the SA content (% mol/mol) and the pHwas varied in a series of 15 samples as detailed in Table 3. For thesesamples, the microdroplet and TRIS concentrations were kept constant atapprox. 4 µl/mL and 5 mM.

TABLE 3 Variance in C2 component characteristics investigated in Study AC2 sample # Microdroplet mean diam. (µm) SA (%) pH 1 1.0 1.5 7.1 2 1.03.5 7.1 3 1.0 5.5 7.1 4 1.8 1.5 6.4 5 1.8 3.5 6.4 6 1.8 1.5 7.1 7 1.85.5 7.1 8 2.4 3.5 6.4 9 2.4 5.5 6.4 10 2.4 3.5 7.1 11 2.4 5.5 7.1 12 3.13.5 6.4 13 3.1 5.5 6.4 14 3.1 3.5 7.1 15 3.1 5.5 7.1

In Study B the microdroplet and TRIS concentration and the microdropletdiameter was varied in a series of 15 samples. In addition, one samplewas thermally degraded by 3 months storage at 40° C. C2 samplesinvestigated are detailed in Table 4. For these samples the pH was keptconstant at 6.2 and the SA content was kept constant at 3%.

TABLE 4 Variance in C2 component characteristics investigated in Study BC2 sample # Microdroplet conc. (µl/mL) Microdroplet mean diam TRIS conc.(mM) 1 4.2 2.1 1 2 4.0 2.1 10 3 3.7 2.1 5 4 3.1 2.0 10 5 2.9 2.0 1 6 2.92.0 10 7 3.0 2.1 1 8 2.6 2.0 10 9 2.8 2.0 1 10 3.6 2.1 5 11 3.7 2.4 5 123.9 2.4 10 13¹ 5.8 1.9 5 14 5.8 2.3 5 15 2.8 2.6 10 1 Sample # 13 wasstored 3 months at 40° C. before use.

E2-3 In-Vitro Characterization

All samples detailed in Tables 3 and 4 where used to prepare andcharacterize DP as detailed in E1. For all samples the content and sizeof clusters was determined by FPIA analysis and the content and size ofactivated bubbles was determined by Sonometry. In addition, for samplesdetailed in Table 3, the Reactivity was measured by Coulter counting.

E2-4 In-Vivo Procedures

For both studies, the following in-vivo procedures were applied.

Animal Handling:

The animal (mongrel or mixed breed dog) arrived on the morning of theexperiment day. There was no acclimatization. Anesthesia was inducedwith pentobarbital (12 - 25 mg kg⁻¹ i.v.) and fentanyl (1.5 - 2.5 µgkg⁻¹) and an endotracheal tube was inserted. The animal was transferredto the operating table and was put on volume-controlled mechanical roomair ventilation (New England mod. 101 Large Animal Ventilator). Whenrequired, O₂-enriched air might be given during some time periods,however not in any of the time intervals from 10 minutes before to 11minutes after test substance injections.

Anesthesia:

The animal was kept in general anaesthesia by a continuous i.v. infusionof fentanyl (20 µg kg⁻¹ h⁻ ¹) controlled by a syringe infusion pump(IVAC model P2000), and pentobarbital (10 mg kg⁻¹ h⁻¹) by drip line. Therate of anaesthetics administered might be adjusted somewhat from thenominal value to assure a constant depth of anaesthesia. The depth ofanaesthesia was monitored by physiological recordings (heart rate, bloodpressure) and by general observation of the animal (signs of muscularactivity, breathing efforts, reflexes).

Body Temperature:

The body temperature was kept constant at 38° by a Harvard homeothermicfeedback control unit.

Surgery and Instrumentation:

A Swann-Ganz catheter for pressure measurements was inserted into thepulmonary artery via the femoral vein and a groin incision. A systemicarterial pressure transducer catheter was inserted into the femoralartery by the same incision. A mid-line sternotomy was performed, andthe anterior pericardium was removed. The heart was suspended in apericardial cradle to avoid compression of the atria and veins. A 0.8 mmVenflon™ cannula was inserted in the right cephalic vein proximal to theelbow joint for injections of test substances.

Physiological Monitoring:

Arterial and pulmonary artery pressure was measured by SensoNor 840transducers (Sensonor AS, Horten, Norway) connected to custom-madedrift-compensated bridge amplifiers (MAX 420, Maxim Integrated Products,Sunnyvale CA). The amplifier outputs are sampled at 500 Hz and fed to a8-channel 12-bit ADC card (CIO-DAS 08, Computer Boards) for furtherprocessing by PC software (Turbo Pascal 5.0, Borland International).Inhaled and exhaled content of O₂ and CO₂ will be continuously monitored(Capnomac Ultima Respiratory Gas Analyzer) but will not be recorded.

The following variables are calculated, displayed and recorded for eachheartbeat: a) Systolic, diastolic and true mean systemic arterialpressure (SAP), b) True mean pulmonary arterial pressure (PAP) and c)Instantaneous heart rate derived from automated (by software) ECG r-wavedetection

Imaging:

A midline, mid-papillary short axis view of the heart was imaged by anATL HDI-5000 scanner. A P3-2 transducer was used, the scanner wasoperated in conventional fundamental B-mode with two focal zones, at thehighest frame rate and maximum output power (MI ≈ 1.0). A 30 mm softsilicone rubber pad was used between the transducer surface and theepicardium. All material interfaces are covered by water-based acousticcontact gel.

The depth of the image was adjusted to the smallest value that willinclude the whole heart. A dynamic range of 50 dB was used. A pair ofdigital images from end-diastole and end-systole was stored at eachspecified point in time. The scanner was left continuously running,except brief periods of cine-loop recalls for storing the images.Digital images are transferred to magnetooptical disk after completionof the experimental session. A PAL VHS video recording of the screen wasperformed to document the procedures. The identity of the animal and allinjections (injection number, substance and dose) should be annotated onthe screen.

Injection Techniques and Dosing:

Prior to each injection, a new vial of C1 was reconstituted with 2 mL ofC2. The desired dose of DP (200 µl) was withdrawn and diluted to 2.5 mLwith 50 mg/mL TRIS-buffered mannitol (10 mM, pH 7.4). The doseadministered was equivalent to 10 µl DP/kg b.w, equivalent to nominally0.04 µl pFMCP microdroplet and 0.08 µl HEPS/PFB microbubbles per kg.b.w. Injections are performed via a Venflon™ cannula equipped with arubber membrane port. The cannula and port dead space (about 0.1 mL) wasflushed with 5 mL of isotonic saline immediately after each injection.

Experimental Procedures:

Injections of DP are made via the right cephalic vein, and the resultingmyocardial contrast effect is quantified at 90 seconds, 3, 5, 7 and 11minutes. A baseline reading was performed before each injection. Atleast 20 minutes was allowed between injections to reduce potentialcarry-over effects.

Data Analysis and Reporting:

For each of the specified time points, myocardial contrast effect wasread from a large region of interest in the anterior myocardium (MathLabsoftware), tabulated against time and illustrated graphically. Thecontrast effect at 90 seconds was used as the primary measure of theefficacy for each injection. Contrast intensity values was reported indB and from these values linear enhancement (Greay Scale units, GS) wascalculated.

E2-5 Results From Study A

The results from in-vitro characterization and myocardial enhancementobserved for the 15 compositions investigated are detailed in Table 5.Several important correlations that elucidate the nature andcharacteristics of the system can be extracted from this data.

Most importantly, we find an optimum in the Reactivity vs. enhancementcorrelation, as shown in FIG. 9 . This covariance clearly demonstratethat there exist an optimal balance in the formation of clusters in DP.

Secondly, we find that the size of the clusters formed is also stronglyconnected to the Reactivity of the system, as shown in FIG. 10 . As canbe observed from this figure, only small clusters (i.e. < 5 um) andmedium sized (i.e. 5-10 µm) are formed at relatively low levels ofReactivity (e.g. < 20%). With increasing Reactivity, larger clustersstart to form; at R > approx. 20%, 10-20 µm clusters start to form andat R > approx. 50%, 20-40 µm clusters start to form. When largerclusters form, it is at the expense of smaller and medium sizedclusters; we find a clear optimum in content vs. Reactivity for clusterconcentration < 5 µm and 5-10 µm.

In combination, the results displayed in FIGS. 9 and 10 demonstrate thatformation of larger clusters is detrimental to the efficacy of thecomposition and that the clustering potential must be balancedaccordingly.

Whilst not wishing to be hold to theoretical speculations, possiblereasons for these effects could be 1) that the larger masses containedin larger clusters prevent or reduce the activation efficacy or 2) thatthe larger clusters are retained in the pulmonary circulation after ani.v. injection and hence does not reach the left ventricle where theactivation is performed.

TABLE 5 Results from in-vitro characterization and in-vivo performanceof investigated compositions - Study A (see text) Sampl e Reactivit y(%) Clusters <5 µm (millions/mL ) Clusters 5 to 10 µm (millions/mL )Clusters 10 to 20 µm (millions/mL ) Clusters 20 to 40 µm (millions/mL )Linear enh. (GS) 1 7 27.1 0.1 0.1 0.0 14 2 60 99.7 18.9 1.0 0.1 253 3 8722.6 8.5 4.5 0.6 102 4 21 111.9 0.8 0.0 0.0 91 5 78 69.8 19.1 2.6 0.0144 6 11 45.9 0.0 0.0 0.0 12 7 84 36.5 9.5 4.2 0.3 147 8 49 66.0 15.72.0 0.0 379 9 70 20.5 9.4 4.2 0.2 180 10 21 112.6 3.8 0.1 0.0 165 11 5084.7 14.0 2.3 0.1 309 12 27 91.3 9.7 0.2 0.0 433 13 59 24.9 12.3 3.3 0.1313 14 16 28.4 1.3 0.0 0.0 229 15 28 58.1 11.5 0.6 0.0 286

E2-6 Results From Study B

The results from in-vitro characterization and myocardial enhancementobserved for the 15 compositions investigated are detailed in Table 6.Several important correlations that elucidate the nature andcharacteristics of the system can be extracted from these data.

Correlations between cluster concentration between 5 to 10 µm and peakactivated bubble volume observed in-vitro vs. myocardial enhancementobserved in-vivo are shown in FIG. 11 .

Examples 20 to 27 given in WO99/53963 also sites data for myocardialenhancement in a model identical to the one described in E2-4 and theprocedures applied are identical. In addition, doses in terms of gas andmicrodroplet volume administered per kg b.w. are comparable betweenthese studies; WO99/53963 sites 0.35 µl gas and 0.04 µlmicrodroplets/kg. b.w. whereas in the current example effective doseswere 0.08 µl gas and 0.026 to 0.059 µl microdroplets/kg b.w. Forcomparison then, the range of enhancement observed and cited in Examples20 to 27 in WO99/53963 has been included in FIG. 11 . Enhancement inthese example was given as dB hence, for this comparison linearenhancement values have been re-calculated from the data sited in Table4, Examples 20 to 27, page 63 in WO99/53963 according as; LinearEnhancement = 10^((dB/10))

As can be noted from FIG. 11 , the in-vivo enhancement is wellcorrelated to the two in-vitro parameters, proving their relevance aspredictors for in-vivo performance; i.e. the clusters comprise theactive component in DP. In addition, the maximum value for linearmyocardial enhancement reported in WO/9953963 was 51 versus 693 in thecurrent study. Applying the concept of the present invention then; bypreparing a composition from C1 and C2 prior to administration, henceforming microbubble/microdroplet clusters, opposed to co-injection ofthe two components as taught by WO/9953963, enable a > 10-fold increasein efficacy.

TABLE 6 Results from in-vitro characterization and in-vivo performanceof investigated compositions - Study B (see text) Sample Clusters <5 µm(millions/mL ) Clusters 5 to 10 µm (millions/mL ) Clusters 10 to 20 µm(millions/mL ) Clusters 20 to 40 µm (millions/mL ) Sonom. vol. (µl/mL )Linea r enh. (GS) 1 103.5 11.5 0.5 0.0 1383 369 2 124.1 4.8 0.1 0.0 796277 3 140.6 10.4 0.2 0.1 1256 284 4 83.0 4.3 0.1 0.0 716 234 5 72.0 5.00.3 0.0 627 181 6 37.1 0.6 0.1 0.0 412 130 7 84.1 14.2 0.9 0.0 923 435 888.6 1.8 0.1 0.0 359 188 9 120.8 6.5 0.1 0.0 460 207 10 131.6 13.2 0.50.0 1073 475 11 125.0 26.4 1.4 0.4 1228 453 12 161.2 29.3 1.4 0.0 1443661 13 15.9 0.2 0.0 0.0 83 33 14 105.7 29.6 6.3 0.1 1740 693 15 208.526.3 1.9 0.1 916 489

E2-7 Multivariate Analysis, Target Cluster Size and CircularityDifferentiation

The results for cluster content in the various size classes and in-vivoenhancement, for all data reported in E2-5 and E2-6, allows for astatistical evaluation of the contribution of the various cluster sizeclasses to in-vivo efficacy. For this purpose a multivariate, principlecomponent analysis was performed. The correlation between the content inthe various cluster size classes (X) and enhancement (Y) was determinedby partial least squares regression (PLSR). The PLSR algorithmdiscriminates noise to extract and define true correlations. Thevalidation of PLSR models was performed by applying full crossvalidation (CV). The CV procedure keeps one sample out followed bytesting the precision of the model by estimating (predicting) Y for theexcluded sample and compare with the measured Y. The procedure wasrepeated for each sample, and the number of models was hence equal tothe number of samples. By comparing all models derived by crossvalidation, the significance of X variables were determined byevaluating the variation in regression coefficients originating fromeach model (p=0.05). The final model is developed from all 30 samples.

Model accuracy and reliability was done by comparing predictedenhancement and measured enhancement and reliable models were verifiedby classic statistical quality estimates (r, RMSEC, RMSEP). Theevaluation of additional statistical parameters as model leverage andsample distance to model concluded that no critical outliers influencedthe model solutions. The Unscrambler software v.9.8, Camo ASA, was usedfor statistical analysis.

The results from this analysis are shown in FIG. 12 . As can be observeda statistical significant contribution to myocardial enhancement isfound for clusters < 5 µm and, more strongly, for clusters between 5 to10 µm, corroborating the two-dimensional analysis displayed in FIG. 11 .Cluster between 10 to 20 µm does not contribute significantly to theenhancement, nor does clusters between 20 to 40 µm, the latter are evenindicated to have a negative contribution, corroborating the resultsdisplayed in FIGS. 9 and 10 . The results demonstrate that the formationof larger clusters reduces the functionality of the concept as thisformation depletes the concentration of functional clusters below 10 µm.

The medium Reactivity (R = 46%) sample reported in Table 4 represents DPwith the cluster attributes that should be targeted. FIG. 13 shows thecluster size and circularity distribution of this sample. As can benoted the clusters in this sample are between ~3 to ~10 µm and display acircularity less than 0.9.

E2-8 Conclusions

According to data and discussions detailed in E1 and E2 we have shownthat

-   Formation of microbubble/microdroplet clusters upon combination of    the 1^(st) component and 2^(nd) component, i.e. in the cluster    composition or pharmaceutical composition, is a prerequisite for its    intended functionality in-vivo.-   Targeted cluster attributes in terms of size is less than 10 µm, and    differentiation from free microbubbles/microdroplets can be    designated by a circularity < 0.9.

EXAMPLE 3 (E3) - ACTIVATED BUBBLE SIZE IN-VIVO E3-1 Introduction

In order to study and demonstrate the characteristics of large phaseshift bubbles produced after activation of the microbubble/microdropletclusters present in the composition, a methodology that allows forin-vivo determination of activated bubble size and dynamics in arelevant animal model has been developed.

E3-2 Components and Compositions Investigated

The compositions investigated in this study were as detailed in E1-2.

E3-3 Methodology

Measurement of activated bubble size distribution and yield ofactivation was performed in a dog model. The study was approved by thelocal animal welfare committee. A cannula was placed in the aorta toallow blood flow through an extracorporeal measurement chamber thatperforms the acoustic bubble sizing. Compound was administered by intravenous injection at 10 µl DP/kg. b.w. and activation provided by aclinical ultrasound scanner imaging the cardiac chambers.

In order to provide consistency data, compound was also administered viaa left atrium cannula with acoustic activation in the cannula, thusproviding data that can be directly compared to the same administration(activation in the cannula) into the in-vitro bubble sizing system.

A mathematical model was developed to calculate the volume of activatedbubbles liberated from the measurements performed in the extracorporealmeasurement chamber. Results of the model were validated by injectingactivated bubbles into the left atrium via a cannula, and comparing theresult to the same administration in the in-vitro measurement system.

At least 15 minutes was allowed between each dosing. Injections wereperformed with an 18 G needle through a rubber membrane port on aforelimb Venflon™ i.v. cannula. Due to the low dose levels, all i.v.injections were given after 1:10 dilution of DP with an aqueousmannitol/ TRIS solution. The injections were given in about 5 seconds,followed by a flush of saline. On some occasions, injections wereperformed into the left atrium of the heart via a short polyethylenecatheter, either with or without prior activation of the drug product byultrasound ex vivo. Left atrium injections were slow (20 seconds) tosimulate the temporal dispersion of the bolus during normal lungpassage. Due to the need for diluting the injected sample in order forthe ex vivo ultrasound exposure to penetrate into the fluid, the atrialinjections were further diluted with isotonic saline to a total volumeof 20 mL.

The body temperature was kept constant at 38 degree C by a Harvardhomeothermic feedback control unit (rectal temperature sensorcontrolling an electrical heating blanket). A Swann-Ganz catheter forpressure measurements and monitoring of cardiac output (Baxter VigilanceContinuous Cardiac Output (CCO) monitor) was inserted into the pulmonaryartery via the femoral vein and a groin incision. A systemic arterialpressure transducer catheter was inserted into the femoral artery viathe same incision. A 1.4-mm Venflon™ cannula was inserted in the rightcephalic vein proximal to the elbow joint, for injection of testsubstances. A midline sternotomy was performed, and PEEP was applied tothe respirator outlet when entering the pleural spaces. The anteriorpericardium was removed, and the heart was suspended by suturing the rimof the remaining pericardium to the wound edges. The auricular appendixof the left atrium was cannulated for injections of activated DP,bypassing the pulmonary circulation.

The animal was fully anticoagulated by a single intravenous injection ofHeparin (1000 i. u. / kg body weight) after complete surgical hemostasiswas achieved, and before extracorporal circulation was started. Theextracorporal shunt and its associated tubing were filled with isotonicsaline and all air was evacuated from the system before the connectionsto the carotid and jugular catheters were established.

The pressure inside the acoustic measurement chamber was checked atregular intervals by briefly connecting the pulmonary artery pressuretransducer to a side port on the chamber, keeping the transducer at thesame elevation level as the chamber.

No significant deviations in flow or pressure in the shunt circulationwere observed during the experiments, and no fibrin clot deposits wereobserved inside the shunt devices after the experiments. Thus,anticoagulation was adequate.

A mathematical model of the flow system was developed, in order toestimate the peak concentration in the measurement cell, as a functionof flow rate into the cell, and the activated bubble half-life, andbolus half-life. The flow rate may then be adjusted, by altering theflow resistance, in order to ensure adequate dose to the measurementcell. In addition, a mathematical model to estimate the concentration ofactivated bubbles in the arterial blood from the concentration observedin the measuring cell.

E3- 4 Results

FIG. 14 shows a typical activated bubble concentration-time curve in thearterial blood compartment after correction for cardiac output, flowthrough cell, transit time to cell, and activated bubble lifetime. Onlythe first two minutes are plotted for clarity of display. The x-axis isthe time in seconds and the y-axis is the activated bubble populationgas fraction in arterial blood in parts per million.

Table 7 below shows the volume-weighted mean activated bubble diametersafter i.v. injection measured at arterial conditions (normal arterialblood gas saturation, hydrostatic pressure of about 60 mmHg). The meanvalue of all observations in the table is 21.4 µm.

TABLE 7 Volume weighted, mean activated bubble diameter at arterialconditions Injection # Dog 1 Dog 2 Dog 3 1 19.7 µm 22.2 µm 20.5 µm 221.7 µm 22.3 µm 21.8 µm 3 91.3 µm 21.3 µm 21.6 µm

Table 8 below shows the rates of activated bubble shrinkage at arterialand venous pressure, given as half-life of gas volume fraction decay inthe acoustic measurement chamber. The pressures have been calculatedfrom catheter/ transducer measurements of arterial pressure, andassuming a venous (jugular vein) pressure of zero. Note the faster decayat arterial pressure, this is caused by the elevated partial pressure ofall gases inside the activated bubbles, giving larger gradients foroutward gas diffusion.

TABLE 8 Arterial and venous chamber pressures and half-lives ofactivated bubbles Arterial or Venous side Dog 1 Dog 2 Dog 3 Pressure inchamber Half-life Pressure in chamber Half-life Pressure in chamberHalf-life Arterial 87 mmHg 21 s 87 mmHg 18 s 88 mmHg 21 s Venous 20 mmHg38 s 20 mmHg 33 s 20 mmHg 36 s

The activated bubbles in arterial blood have diameters of 20 - 22microns, well within the predicted range. After injection of thesubstance into the left atrium and activating in the left ventricle theactivated bubbles become slightly larger, 22 - 25 micron in diameter.Verification of correct measurements and calculations in all animals hasbeen obtained by parallel in-vitro analysis with activation of theinjected samples by US irradiation.

E3-5 Conclusions

Example 3 confirm that the composition is activated within the desiredMI range and produces bubble growth and dynamics within the desired sizerange in vivo after intravenous administration.

EXAMPLE 4 (E4) - INTRAVITAL MICROSCOPY ON DEPOSIT NATURE OF ACTIVATEDBUBBLES AND MODELING OF RESPONSE TO US IRRADIATION, COMPARED TO REGULARMICROBUBBLES E4-1 Introduction

In order to further study and demonstrate the characteristics of largebubbles produced after activation in-vivo, a study directly observingindividual activated phase shift bubbles within the microcirculation viamicroscopy of rat mesentery was performed. In addition, to describe thesignificant differences between the large activated bubbles from thecurrent invention and regular US contrast microbubbles e.g. such asSonazoid, a theoretical modelling of the volume response to USinsonation was performed.

E4-2 Components and Compositions Investigated

The compositions investigated in this study were as detailed in E1-2.

E4-3 Methodology

Male Wistar rats were used in the study. The composition wasadministered intravenously at a dose of 1 mL DP/kg b.w. (i.e. 4 µL/kgb.w. microdroplets and 8 µL/kg b.w. microbubbles). General anaesthesiawas administered and maintained with i.v. and i.m. pentbarbital sodium.The rats were intubated, and the tail vein or carotid vein wascannulised for administration of the test formulation. Ultrasound wasapplied to activate the clusters in the mesentery. The abdomen wasopened by means of a vertical midline incision, the rats were thenplaced in the lateral position on a plastic plate incorporating a roundwindow of cover glass, and the exteriorized mesenteries were placed onthe cover glass window. The spread mesenteries were perfused withKrebs-Ringer buffer at 37° C. Ultrasound was applied directly onto theexteriorised mesentery under the objective lens of the microscope. Anultrasound scanner (Elegra; Siemens, Seattle, WA) equipped with a linearprobe (7.5L40) was used for ultrasound exposure. Output power was set atmaximum corresponding to an MI value of 1.2. Sonar gel was appliedbetween ultrasound transducer and chest wall or the mesentery. Imageswere recorded on S-VHS or DV tape for subsequent review.

Simulations of the change in volume of the activated bubbles from thecurrent invention and regular HEPS/PFB microbubbles (C1 reconstitutedwith water) upon insonation was modelled using the nonlinear bubblemodel developed by Lars Hoff and described in Acoustic Characterisationof Contrast Agents for Medical Ultrasound Imaging, Kluwer AcademicPublishers, 2001, Chapter 8. Simulation parameters for activated phaseshift bubble: 8 cycles driving pulse with a MI of 0.2 and frequency of0.5 MHz, in blood, and 30 micron resting diameter. Simulation parametersfor HEPS/PFB microbubbles: 8 cycles driving pulse with a MI of 0.2 andfrequency of 5 MHz, in blood, and 3 micron resting diameter.

E4-5 Results No Ultrasound Activation:

Two animals were used. No large phase shift bubbles were observed in themesentery microcirculation after the 6 injections performed.

Ultrasound Activation:

Three animals were used. Large, activated bubbles were observed afterall injections. Activated bubbles were only observed after applicationof ultrasound. The growth phase of the activated phase shift bubblescould be observed in real-time. The nucleus of the activated bubble grewwithin a few seconds along with micro vessel blood flow obstruction. Noexpansion of the micro vessels was observed. The activated bubblesgradually shrank and intermittently advanced in the micro vessels. Allactivated phase shift bubbles were larger than red blood cells andlodged in the micro vessels and transiently blocked blood flow. Allactivated bubbles were non-spherical and appeared ellipsoidal in shape,forming against a section of the micro vessel.

FIG. 15 shows video frames of an activated phase shift bubble in themesentery at; (top left) 17 seconds post-injection in a micro vessel,blocking blood flow; (top right) at 5 minutes and 19 seconds; (bottomright) at 5 minutes and 45 seconds, respectively. The activated phaseshift bubble (indicated by the arrow) gradually shrinks and advances inthe micro vessel by intermittent lodging and dislodging, before itclears completely. FIG. 15 (bottom left) shows a 5 times schematic zoomof the dashed rectangular box indicated in the image (top left). Theoutline of the phase shift bubble is shown (A) with a 20 micron scalebar shown in C. Assuming a cylindrical bubble, the length is measured tobe 30 micron and width 13 microns giving a volume of 3982 cubic micronsequivalent to a 20 micron diameter spherical bubble, in excellentagreement with the results detailed in E3. A HEPS/PFB microbubble (B) isshown to scale. These regular US contrast microbubbles are clearlyfree-flowing in the microvasculature and not in contact with theendothelial wall.

Simulation of Bubble Dynamics When Exposed to Ultrasound Field:

Results are shown in FIG. 16 . The phase shift bubble has a minimumdiameter 22.8 and maximum diameter 36.4 microns when oscillating inresponse to the driving ultrasound field. The HEPS/PFB microbubble has aminimum diameter 2.3 and maximum diameter 4.1 microns when oscillatingin response to the driving ultrasound field. The absolute volume changesinduced for the phase shift bubbles is approx. three orders of magnitudegreater than the HEPS/PFB microbubble.

E4-5 Conclusions

Activated phase shift bubbles with a size of approximately 20 µm wereobserved when ultrasound activation was applied. No activated phaseshift bubbles were observed when ultrasound activation was not applied.The activated phase shift bubbles were transiently (5-19 minutes)deposited in the microcirculation but dislodged as their size decreased.

Simulation of the volume changes of a phase shift bubble during USinsonation shows a three orders of magnitude greater response than aHEPS/PFB microbubble, demonstrating the orders of magnitude greatermechanical work exerted on the tissue by the phase shift bubble.

EXAMPLE 5 (E5) - FORMULATION STUDIES E5-1 Introduction

As apparent from E1, the inventors have deliberately chosen to focusformulation studies on variance in C2. This in order to study generaleffects on the ability to form clusters upon preparation of DP, andhence to obtain control with cluster characteristics and elucidate theirimportance. It is reasonable to assume that the general formulationaspects/effects shown in E1 apply for a wide variety ofmicrobubble/microdroplet formulation systems. In order to show this, sixcommercially available microbubble formulations have been tested forpreparation of the cluster composition and subsequent activation.

In addition, two important aspects of the invention; the stability ofthe pharmaceutical preparation in terms of avoiding spontaneousactivation (as noted in WO99/53963) and the ability to load themicrodroplets with a therapeutic agent are elucidated in the currentexample.

E5-2 Cluster Compositions From Commercially Available MicrobubbleFormulations

In order to show that the concept of the current invention is applicableto a wide variety of microbubble formulations, DP made from C2 asdetailed in E1-2 and six commercially available microbubble products asC1, were tested for cluster content by microscopy/image analysis andactivated bubble volume and diameter by Sonometry. The microbubblecomponents investigated as C1 are detailed in Table 9 together withvendors, composition of gas core, stabilizing membrane andpharmaceutical form.

TABLE 9 Commercially available microbubble formulations tested as C1Product (C1) Vendor Gas core Stabilizing membrane Form Sonazoid GEHealthcare PFB HEPS-Na Lyophilized Optison GE Healthcare PFP Humanalbumin Aqueous dispersion Sonovue Bracco Spa SF₆ DSPC, DPPG-Na,palmitic acid, PEG4000 Lyophilized Definity Lanteus Medical Imaging Inc.PFP DPPA, DPPC, PEG5000-DPPE, hexadecanoic acid Aqueous dispersionMicromarker VisualSonics Inc. PFB, N₂ Phospholipids, polyethylenglycol,fatty acid¹ Lyophilized PolySon L Miltenyi Biotec GmbH Air Inert,organig polymer¹ Aqueous dispersion ¹ Exact chemical composition is notdisclosed by the manufacturer.

For lyophilized forms, preparation of cluster compositions was performedas detailed in E1-2, reconstituting the C1 with a volume of C2 asdetailed in the package insert of each formulation (2 mL for Sonazoid, 5mL for Sonovue and 0.7 mL for Micromarker). For Optison and Definity themicrobubbles in a product vial was isolated by removal of infranatantafter segregation of the microbubbles and the cluster composition wasprepared by adding the same volume of C2 to the vial beforehomogenisation. For Polyson L, 0.5 mL of homogenized C1 was mixed with0.5 mL of C2.

Results for cluster content and activated bubble volume and diameter inthe various cluster compositions are stated in Table 10. The C1component detailed in E1-2 has the same formulation and form as thecommercial contrast agent Sonazoid, hence it would be expected thatthese two agents, when used as C1, would generate a cluster compositionswith similar characteristics; as confirmed by the results stated inTable 11. Of the other 5 commercial microbubble product investigated allbut Definity yield a cluster composition with significant amounts ofclusters which, upon US irradiation, were activated and displayed asignificant activated bubble volume. Micromarker, Optison, Sonovue andPolyson, although displaying a strong variance in the chemicalcomposition of the gas core and the stabilizing membrane, showcharacteristics for their respective cluster compositions which arecomparable to those prepared with Sonazoid and C1 as detailed in E1-2.Whilst not wishing to be bound by theoretical considerations it ispossible that the reason why the Definity microbubbles does not formclusters with the microdroplet in C2 is the use of the PEG-DDPEcomponent in the stabilizing membrane. This component is likely tocreate a thick, dens layer of water surrounding the microbubble, thusscreening the electrostatic attraction to the microdroplets of C2. Anadditional finding from this study, when using a 50X objective with ameasuring range of 0.5 to 40 µm, was the observation of a significantamount of clusters smaller than 3 µm in the cluster compositions madewith Sonovue and PolySon L. These microbubble agents contain asignificant amount of small microbubbles compared to C1 as detailed inE1-2, Sonazoid or Optison. In the cluster compositions made with Sonovueand PolySon a significant fraction of clusters formed from ~1 µmmicrobubbles and ~1 µm microdroplets was observed, and these apparentlycontributed to the activated bubble volume after US irradiation.Clusters in the size range 1-10 µm should hence be regarded asfunctional under the current invention.

TABLE 10 Cluster content and activated bubble volume for clustercompositions prepared using various commercially available microbubbleformulations as C1 Product (C1) Cluster cone. (milions/mL) Activatedbubble volume (µL/µL) Activated bubble diameter (µm) Sonazoid 45 293 40Optison 23 232 48 Sonovue 32 226 50 Definity 0 0 NA Micromarker 41 29348 PolySon L 23 167 48

These results reported above demonstrate that the concept of the currentinvention is applicable to a wide variety of C1 formulations, both withregards to the composition of the gas core and with regards to thecomposition of the stabilizing membrane.

E5-3 Spontaneous Activation and US Activation

The basic nature of the formulation is directed towards adestabilisation of the system i.e. the US induced generation of largephase shift bubbles from the combination of microbubbles andmicrodroplets. This destabilisation must occur in a controlled manner,in-vivo and at the target site (pathology), and spontaneous growth(activation) upon preparation of DP, or immediately after administration(i.e. in the absence of insonation) is detrimental to the functionalityof the invention. WO99/53963 only explore co-administration of the twocomponents but notes that, if the components are mixed prior toadministration, avoiding such spontaneous activation of the system islikely to require storage at elevated pressure or low temperature aftercombination of C1 and C2. The inventors has tried to eliminate theseobviously cumbersome and limiting needs to provide a formulation that isstable at ambient conditions. As noted inWO99/53963, based on atheoretical evaluation, it is likely that spontaneous activation is afunction of the boiling point (b.p.) of the oil phase and its vapourpressure (v.p). However, the authors of this patent does not identifiedthe possibility that the water solubility of the oil phase may be aneven more important contributor to spontaneous destabilisation andbubble growth upon combining C1 and C2. To elucidate these relationshipsand to provide a solution to this problem, a number of microdropletphase components (fluorocarbon oils), with a wide range of b.p., v.p.and water solubility, was screened and used for manufacture of C2. Thesesamples were then combined with C1 and assessed for content ofspontaneously activated and US activated bubbles. Manufacturing andanalysis of these samples are described in the following.

641 mg distearoylphosphateidylcholine (DSPC) and 73 mg1,2-distearoyl-3-(trimethylammonio) propane chloride (DSTAP) wereweighed into a 250 mL round bottom flask and 50 mL chloroform was added.The sample was heated under hot tap water until a clear solution wasobtained. The chloroform was removed by evaporation to dryness on arotary evaporator at 350 mm Hg and 40° C., followed by further drying at50 mm Hg in desiccator over night. Thereafter, 143 mL water was addedand the flask again placed on a rotary evaporator and the lipids wererehydrated by full rotational speed and 80° C. water bath temperaturefor 25 minutes. The samples were placed in refrigerator over night. Thelipid dispersion was transferred to a suitable vial and stored inrefrigerator until use.

Emulsions were prepared by transferring aliquots of 1 mL of the coldlipid dispersion to 2 mL chromatography vials. To each of seven vialswas added 100 µL of the fluorocarbon oils as detailed in Table 11. Thechromatography vials were shaken on a CapMix (Espe, GmbH) for 75seconds. The vials were immediately cooled in ice, pooled and kept colduntil use. Coulter counter analysis was performed to determine thevolume concentration of the microdroplets and the emulsions were thendiluted with water to 10 µl/mL disperse phase.

C1 (as detailed in E1-2) was reconstituted in 2 mL of water and mixedtogether with the C2 samples prepared in a 10 mL tube to a ratio of 10:1and shaken carefully by hand. The mixture was then diluted with 7 mLwater. The samples were evaluated for spontaneously activated and USactivated bubbles by microscopy in manual version of the methodologydescribed in E1-4. One mL of this solution was transferred to amicroscope cell where the temperature was stabilised to 37° C. after 2minutes. The cell was set up so that US sonication, using an ATL 3-2transducer with a center frequency of 2.25 MHz, could be applied to thesample. At 200X magnification the entire cell area was scanned and thecontent of large (> ~ 15 µm), spontaneously activated bubbles was semiquantitatively assessed by visual inspection. For each sample, a scorein the range of 0 to 2 was given, where 0 designate “no or very fewlarge phase shift bubbles observed”, 1 designate “medium number of largephase shift bubbles observed” and 2 designate “large number of largephase shift bubbles observed”. The sample was then insonated for 5 s ata nominal MI of 0.8 and the content of large, US activated bubbles werecounted and scored in the same manner. The results from this study isdetailed in Table 11 together with the physicochemical characteristicsof the compounds investigated.

TABLE 11 Compounds investigated and their physicochemicalcharacteristics; boiling point (b.p., °C), vapour pressure (v.p., torr)at 20° C. and water solubility (logarithm of Molar solubility). Comparedto results from assessment of the amount of spontaneously and USactivated bubbles. Score 0 = no or very limited, Score 1 = medium andScore 2 = high Compound b.p. v.p. Log W_(sol) Spont. Act. US Act.Methyl-1.1.2.2-tetrafluoroethyl 34 842.4 -1.4 2 02.2.3.3.3-Pentafluroropropyl methyl 46 559.6 -2.0 1 0Perfluorodimethylcyclobutane 45 579 -5.7 0 1 Perfluoromethylcyclopentane48 522.7 -5.5 0 1 2H.3H-perfluoropentane 53.6 431.8 -3.2 1 01.1.2.3.3.3-Hexafluoropropyl 54.5 418.8 -2.5 2 0 Perfluorohexane 59359.4 -6.7 0 1 1H.1H.2H-perfluoro-1-hexane 59.5 353.1 -4.6 1 11H-perfluorohexane 71 238.6 -5.3 1 1 Perfluoroheptane 82.5 161.2 -7.5 02

The data sited in Table 11 surprisingly shows that the level ofspontaneous activation is not significantly correlated to b.p. or v.p.,but strongly so to the water solubility of the oil component, as was thelevel US activation. FIG. 17 shows water solubility vs. spontaneous andUS activation score. As can be noted a marked decrease in the formationof spontaneously activated bubbles is observed with decreasing watersolubility, whereas a marked increase in the level of US activatedbubbles are observed with decreasing water solubility. These resultsdemonstrate that DP can be stabilised against spontaneous activation byusing oil components for C2 with a water solubility below approximately1·10⁻⁵ M and also that the level of US activation benefits from a watersolubility below this level. Note, however, as shown in E5-4, asignificant fraction of the emulsion microdroplet component can becomprised by components (e.g. co-solvents added for increased drugloading) with a higher water solubility without leading to increasedspontaneous activation or decreased in US induced activation.

E5-4 Drug Loading and Co-Solvents

In one aspect of the invention, a therapeutic compound is added to themicrodroplet oil phase for release at targeted site in vivo uponactivation. In order to elucidate concepts to achieve such loading aseries of formulation studies were performed. These are brieflysummarized in the following.

Based on screening studies of the various components reported in E5-3,with additional responses such as ease of emulsification, stability ofemulsions, availability, quality etc., perfluoromethylcyclopentane(pFMCP) was selected as the primary oil component for manufacture of C2,with a distearoylphosphateidylcholine (DSPC) stabilising membrane addedstearlyamine (SA) for positive surface charge. As a starting-point forthe study on drug loading, a theoretical evaluation of solubility ofdifferent solutes (drugs and chromophores for optical imaging) in pFMCPand a range of other oil components was performed. This evaluation wasperformed using a state-of-the-art software for assessment ofsolvent-solute compatibility; Hansen solubility parameters, HSPiP v.4(Steven Abbott TCNF Ltd.). The HSPiP analysis calculates three basicproperties relating to compatibility between substances; Polarity,Dispersion and Hydrogen binding and a distance in this three dimensionalspace between e.g. a solvent and a solute; the Hansen distance (H_(d)).The closer the solvent and solute are in this space, the (relatively)better the solubility of the solute in the solvent. Hansen theorypredicts that a H_(d) < 8 represents a soluble “solute in solvent” pair,8 < H_(d) < 12 represent partial solubility and H_(d) > 12 representsnon-solubility. This analysis was performed for 1) a series of solvents,selected based on b.p. < 65° C., water solubility < 0.1 M and probablebiocompatibility (toxicity), with a large span in Hansen parameters and2) a series of targeted solutes; chemotherapeutic drugs and moleculessuitable for optical imaging. Based on the stated solvent selectioncriteria, preferred solvents were all partially halogenatedhydrocarbons. The miscibility between the solvent and the solubility ofthe solutes in one of the solvents were experimentally determined. Theresults from this study are stated in Table 12. In addition to the datastated there, it was found that chlorotrifluoropropane (CltFPr) anddicholorodifluoroethane (dCldFEt) was completely miscible in pFMCP,dicholormethane (dClMe) and tricholormethane (tClMe).

TABLE 12 Physicochemical properties of solvents; boiling point (b.p.),vapour pressure and water solubility (logarithm of Molar solubility andtarget molecules, Hansen distance (Hd) from pFMCP and tCIMe andmiscibility/solubility in tCIMe (see text) Solvent/Target b.p. LogW_(sol) H_(d) PFMPC H_(d) tClMe Miscibility (%) and Solubility (mg/mL)in tClMe pFMCP 46 -5.5 - 11.3 ~ 10% CltFPr 51 -2.4 7.1 6.2 100%(Complete) dCIMe 40 -0 8 11.9 47 100% (Complete) dCldFEt 55 -2.3 8.7 4.3100% (Complete) tClMe 61 -1.2 11.3 - - Nile Red (dve - - 15.9 6.5 ~ 50DiR (dve molecule)¹ - - 20.3 10.4 ~ 50 Irinotecane - - 15.0 6.6 ~ 350SN38 - - 18.5 0.6 ~0 Paclitaxel - - 16.7 11.4 ~ 350 Docetaxel - - 23.116.1 ~20 Doxorubicin - - 21.2 15.5 ~ 1 Hesperadin - - 20.7 12.1 ~7.5Idealsib - - 17.5 9.5 ~ 10 Gemcitabine - - 23.1 16.1 ~0 Tosacertib - -24.6 16.1 ~0 ZM44739² - - 17.1 8.4 ~ 1 Afatinib - - 18.1 9.5 ~ 100 ¹DiIC₁₈(7) (1,1′-Dioctadecyl-3,3,3′,3′-TetramethylindotricarbocyanineIodide (Life Tech. Ltd) ² Experimental aurora kinase inhibitor(Selleckchem Ltd.)

As can be noted from these results, whereas the calculated H_(d) fitsreasonably with the predicted miscibility between solvents, it is not agood predictor of the absolute solubility of the various targetmolecules in tClMe. This shows that the Hansen analysis is primarily atool for the relative solubility of a given molecule in various solventsystem, and cannot be used to estimate absolute solubility of variouscompounds in various solvent systems.

For seven of the substances sited in Table 12, the measured solubilityin tClMe was also correlated to literature values for LogP and LogS.Whereas this analysis indicated, as expected, that the solubility intClMe is a function of the lipophilicity, these characteristics couldnot predict absolute solubility. Substances with a LogP < 0.9 and alogS > -2.7 did show no or very low solubility, but for substances witha LogP range of 3.2 to 3.9 the solubility varied from 50 to 350 mg/mLwith no covariance to LogP, and for substances with a LogS range of -3.7to -5.2 the solubility varied from 20 to 350 mg/mL with no covariance toLogS.

These evaluations show that, whereas lipophilic substances arepreferred, the compatibility between any specific therapeutic agent andthe invention needs to be tested experimentally.

None of the target molecules in Table 12 displayed any measurablesolubility in pFMCP, hence the use of a co-solvent in order to achieve afunctional loading capacity is necessary. As the miscibility of d- andt-ClMe in pFMCP is only some 10%, a “solvent ladder” construction, i.e.the use of a third solvent between tClMe and pFMCP in the Hansen space,is indicated. Based on these considerations a 1:1:1 (by volume) mix ofpFMCP, ClrFPr and pFMCP was selected for further studies on C2 loadedwith therapeutic or optical imaging compounds.

The solubility of Nile Red (NR), DiR and Paclitaxel (Ptx) was evaluatedin the 1:1:1 mixture of pFMCP, ClrFPr and pFMCP and found to > 5mg/mL, > 10 mg/mL and > 25 mg/mL, respectively. In addition, a 1:1:2mixture of said three solvents loaded with Ptx was explored. Thesolubility of Ptx in this solvent mixture was > 50 mg/mL, showing thatthe loading capacity can be substantially increased by changing thecomposition of the oil phase. C2 with a 1:1:1 mixture of thesecomponents was manufactured as detailed below.

A lipid dispersion containing was made by weighting out 250 mg of DSPCwith 3% mol/mol SA to 50 mL of water in a 100 mL round bottom flask,hydrated for 30 minutes at 80° C. and allowed to cool. X mg substance (Xbeing 5, 10 and 25 mg for NR, DiR and Ptx, respectively) was weightedout and dissolve in 333 µL tClMe (solution A). 333 µl of solution A wasdiluted with 333 µl CltFPr + 333 µL pFMCP (solution B). 900 µl lipiddispersion was added to a 1.5 mL centrifuge tube. 100 µl of solution Bwas added to the lipid dispersion in the centrifuge tube. Emulsificationwas achieved using a ZoneRay® Dental HL-AH G7 Amalgamator at 3200 rpmfor 20s. The resulting emulsion was centrifuged for 5 min at 25 g. Aftercentrifugation, the microdroplets formed a defined sediment layer. Thesupernatant, containing excess lipid vesicles, was carefully removed, anequivalent volume of 5 mM TRIS in water was added and the microdropletsredispersed by manual shaking. A Coulter analysis was performed andbased on the detected volume concentration of microdroplets the emulsionwas diluted in 5 mM to 3 µl microdroplets/mL.

The resulting samples of C2 was assessed by microscopy and Coulteranalysis. FIG. 18 shows micrographs from the microscopy evaluation. Forall samples, stable emulsions with microdroplet sizes in the targetedrange 1 to 5 µm was observed. In addition, the loaded substances areclearly contained in a dissolved state within the microdroplets; noextra-vesicle material is observed and the microdroplets are clear andhomogeneously coloured with no sign of precipitation. For all samples,the Coulter analysis showed approx. 3 µl microdroplets/mL with a mediansize of approx. 3 µm.

Together with C1 as detailed in E1-2, these C2 samples where then usedfor preparation of DP which was assessed for Reactivity by Coultercounting, clustering by microscopy and activated bubble size and volumeby Sonometry. The observed Reactivity for all samples was in the rangeof 40-70%, microscopy confirmed the presence of clusters, but showed noevidence of spontaneous activation, the activated bubble volume was inthe range of 100-200 µl/µl microdroplets and the activated bubble sizewas in the range of 42-48 µm. These results demonstrate;

-   That the microdroplet oil phase can comprise a range of solvents in    order to obtain an acceptable drug loading capacity. For this,    partially halogenated hydrocarbons are particularly useful.-   That a significant fraction (e.g. > 60% v/v) of the solvents can    have a significantly higher water solubility (e.g. < 1·10⁻¹ M) than    indicated from E5-2 (< 1·10⁻⁵ M).-   That these formulations retain the critical attributes of the    concept in the formation of clusters in the cluster composition,    their ability to be activated upon insonation and the lack of    spontaneous activation.

C2 samples loaded with DiR as described above were used for assessmentof delivery in-vivo (see E8). C2 samples loaded with NR as describedabove was used for assessment of the expression of the loaded substanceupon activation (see E6).

EXAMPLE 6 (E6) - EXPRESSION OF LOADED SUBSTANCE UPON ACTIVATION E6-1Introduction

In order to investigate how a molecular substance, loaded into themicrodroplets of C2, will be expressed after activation of the clustercomposition, a fluorescence microscopy study was performed. A clustercomposition where the microdroplets in C2 had been loaded with Nile Red(NR) dye was activated and studied by fluorescence microscopy.

E6-2 Compounds and Procedures

C2 loaded with 5 mg/mL Nile Red dye, as detailed in E5-4, was used toprepare a cluster composition as detailed in E1-2. The clustercomposition was diluted in water, placed in a microscopy well andactivated using a Vscan US scanner (GE Healthcare).

Images of the activated cluster composition were acquired using a LeicaTCS SP8 confocal microscope. The objective used was a HCX IRAPO L 25Xwater immersion objective with a numerical aperture of 0.95. Thefluorescent dye was excited at 539 nm by a tunable white light laser.Emission in the range 570 - 670 nm was detected by a hybrid detector(HyD). The laser speed used was 400 Hz and pinhole diameter was set to 1AU. Transmission images were acquired simultaneously in anotherdetector, which could be overlaid with the fluorescence images.Intersections and 3D images of the sample were acquired by moving theobjective nosepiece stepwise in the z-direction.

E6-3 Results

FIG. 19 shows a fluorescence micrograph of an intersection of phaseshift bubbles after activation of DP where the C2 microdroplet componentwas loaded with 5 mg NR/mL.

As can be noted from this figure, after activation the loaded NR ispresented at the liquid/gas interface. After activation in vivo, ifloaded with therapeutic substance, such substance would be in closecontact with the endothelial wall and hence accessible forextravasation.

EXAMPLE 7 (E7) - DEPOSITION OF ACTIVATED BUBBLES IN TUMORS E7-1Introduction

In order to further study and demonstrate the characteristics of largebubbles produced after activation in a tumour model, a study imagingactivated phase shift bubbles in subcutaneous prostate cancer PC-3tumour xenografts in a murine model was performed, demonstrating thedeposit nature and marked difference in contrast enhancement kineticsfrom free flowing HEPS/PFB microbubbles.

E7-2 Components and Compositions Investigated

The compositions investigated in this study were as detailed in E1-2.

E7-3 Methodology

16 Female Balb/c nude mice were used. Before tumour implantation, micewere weighted, anesthetized with isoflurane, and ear marked. 100 µL cellsuspension containing 3·10⁶ PC-3 cells were slowly injectedsubcutaneously on the lateral side of the left hind leg between the hipand the knee.

The mice were administered surgical anesthesia by subcutaneous injectionof a mix of Fentanyl (0.05 mg/kg), Midazolam (5 mg/kg), and Medetomidine(0.5 mg/kg). An intravenous cannula (BD NeoflonTM 24 GA) was placed inthe tail vein. Patency was verified by injection of a slight amount (~20µL) of 0.9% sodium chloride for injection after which a small amount of(~10 µL) heparin (10 U/mL) was injection to prevent clotting. The hub ofthe cannula was filled with 0.9% sodium chloride for injection toeliminate any dead space and closed with a cap. The cannula was securedto the tail with surgical tape.

Three commercial ultrasound imaging systems were used. The tumour wasimaged for all experiments with a high frequency small animal imagingsystem Vevo 2100 (VisualSonic Inc.) with a MS250 transducer (16-18 MHz).The cluster composition was activated in-vivo either with a Vivid E-9clinical imaging system (GE Healthcare) using a 2 MHz imaging probe withMI setting of 0.28, or a Vscan 1.2 clinical imaging system (GEHealthcare) with a 2 MHz imaging probe with an nominal MI setting of0.8.

The 16 animals were split into 4 groups of 4 animals in each group. Theactivation system and dose for the groups are stated in Table 13.

TABLE 13 Groups investigated with activation procedure and dose GroupActivation Dose 1 Vivid E9. MI 0.28 1.5 µL/kg pFMCP + 4 µL/kg HEPS/PFB 2Vscan. MI 0.8 1.5 µL/kg pFMCP + 4 µL/kg HEPS/PFB 3 Vivid F9. MI 0.28 6µL/kg pFMCP + 16 µL/kg HEPS/PFB 4 Vscan. MI 0.8 6 µL/kg pFMCP + 16 µL/kgHEPS/PFB

The prepared mice were place on a handling table (with temperaturecontrol set to 37° C.), on its right side. The left leg was liftedhorizontally, supported by a piece of cloth and fixated with surgicaltape. Ultrasound gel was richly applied unto the tumour and awater-bath-bag was placed on top of the tumour. Imaging was performedwith the Visualsonics Vevo 2100 imaging system with the transducerplaced in the water bath with the imaging transducer held in a fixedscan plane. Activation of the cluster composition was performed with anadditional transducer (either the Vivid E9 or Vscan) for 75 secondsstarting from the time of injection of the cluster composition.

Activation of the cluster composition produces contrast echoes whichremain stationary in the ultrasound image for several minutes. Thenumber of stationary contrast signals was counted per unit area of thetumour imaged in the scan plane. Assuming a scan plane thickness of 0.2mm the number of phase shift echoes per unit volume of tumour wasderived.

E7-4 Results

The tumour is hypo echogenic in the ultrasound image. Typical tumourimages are shown in FIG. 20 for both pre injection of the clustercomposition (left image) and post injection and activation (rightimage), showing the presence of the stationary phase shift echoes postactivation (right image). The estimated number of stationary phase shiftcontrast echoes per mL of tumour tissue is shown in Table 14. Alltumours showed deposition of stationary contrast echoes. These echoesare termed stationary as they remain static in the ultrasound images forseveral minutes. Application of a burst sequence from the Vevo 2100imaging system, which is designed to destroy regular contrastmicrobubbles such as HEPS/PFB does not destroy the stationary echoes.This is consistent with theory that predicts that the phase shiftbubbles are not destroyed by burst sequences due to their larger size,and confirms that the stationary contrast echoes are not produced by theHEPS/PFB microbubbles in the composition. FIG. 21 shows the typicalintegrated contrast enhancement kinetics in the tumour region from thephase shift bubbles (labelled A in the figure) compared to an equivalentdose of HEPS/PFB microbubbles only (labelled B in the figure). Thisdemonstrates the difference in kinetics of phase shift bubbles comparedto HEPS/PFB microbubbles, i.e. deposit vs. free flowing nature. The freeflowing HEPS/PFB microbubbles enhancement is much more transient. Thecontrast from the phase shift bubbles shows stationary echoes that aredeposited in the tumour and remain for several minutes.

TABLE 14 Estimated mean number of phase shift stationary echoes per mLtumour tissue, and standard deviation (SD) Group Mean number per SD 1(MI 0.28. low dose) 3685 2188 2 (MI 0.9. low dose) 6675 1701 1 (MI 0.28.high dose) 10705 6394 4 (MT 0.9_(.) high dose) 12597 7884

A two-way analysis of variance was applied for dose and type ofactivation (Vivid E9 or Vscan). There is a statistically significantdifference for dose with p=0.024, and an insignificant difference foractivation transducer, p=0.352.

E7-5 Conclusions

The cluster composition was activated with two different clinicalimaging systems, a Vivid E9 with a 2 MHz probe and MI of 0.28, and aVscan with a 2 MHz probe and MI of 0.8, and tumours imaged with a highfrequency (16-18 MHz) small animal ultrasound imaging system. Allprocedures produced stationary contrast echoes in the tumours. Thesecontrast echoes remain stationary in the ultrasound image for severalminutes as opposed to the transient contrast echoes from HEPS/PFBmicrobubbles. They are not destroyed with burst imaging sequencesdesigned to destroy HEPS/PFB microbubbles. These observations areconsistent with phase shift bubble deposition in the tumour tissue. Astatistically significant dose response was observed (p=0.24) and theamount of deposition was not statistically different when activated withthe different clinical imaging systems with MI of 0.28 and 0.8(p=0.352).

EXAMPLE 8 (E8) - DELIVERY OF CO-INJECTED OR LOADED SUBSTANCES TO TUMORSE8-1 Introduction

In order to demonstrate the ability of the current invention to enhancedelivery of molecules in-vivo, studies in a mouse PC-3 xenograph tumourmodel were performed. Three model systems were explored; co-injection ofDP and Evans Blue dye: co-injection of DP and Licor CW800 EPR agent: andinjection of DP where a DiR-dye had been loaded into the microdropletcomponent (C2). Evans blue is a fluorescent dye that binds to albuminprotein when injected i.v. Under physiologic conditions, the endotheliumis impermeable to albumin, and Evans blue bound albumin remains confinedwithin blood vessels. Thus, Evans blue is often used as a model compoundin drug delivery studies [Bohmer et al., J Controlled Release, 148,Issue 1, 2010, pp. 18-24]. Enhanced permeability and retention (EPR) isa common characteristic of tumour vasculature. The vascular endotheliumin the tumour microenvironment is often discontinuous, allowingmolecules to diffuse into the surrounding tumour tissue. Thecommercially available (LiCor Biosciences Inc.) IR dye 800CW PEGcontrast agent (25-60 kDa) is a non-specific imaging agent intendedaccumulate in tumours due to the EPR effect. DiR dye is a commerciallyavailable (Life Technologies, Thermo Fisher Scientific Inc.) near IRfluorescent, lipophilic carbocyanine DiOCis(7) dye which is weaklyfluorescent in aqueous conditions but highly fluorescent and photostablewhen incorporated into e.g. cell membranes. Thus the standard techniquesof extraction and quantification of Evans Blue in tissue, and opticalimaging with the 800CW PEG and DiR dyes, were employed as modelcompounds for in-vivo demonstration of drug delivery with the currentinvention.

E8-2 Components and Compositions Investigated

The compositions investigated in this study were as detailed in E1-2(co-injection models) and E5-4 (DiR loaded).

E8-3 Methodology

Female Balb/c nude mice were used in the study. Before tumourimplantation, mice were weighted, anesthetized with isoflurane, and earmarked. 100-µl cell suspension containing 3·10⁶ PC-3 cells were slowlyinjected subcutaneously on the lateral side of the left hind leg betweenthe hip and the knee.

The mice were administered surgical anesthesia by subcutaneous injectionof a mix of Fentanyl (0.05 mg/kg), Midazolam (5 mg/kg), and Medetomidine(0.5 mg/kg). An intravenous cannula (BD NeoflonTM 24 GA) was placed inthe tail vein. Patency was verified by injection of a slight amount (~20µL) of 0.9% sodium chloride for injection after which a small amount of(~10 µL) heparin (10U/mL) was injection to prevent clotting. The hub ofthe cannula was filled with 0.9% sodium chloride for injection toeliminate any dead space and closed with a cap. The cannula was securedto the tail with surgical tape.

The hind limb of the mouse was placed in a water bath with two UStransducers poised for insonation of the tumour. Ultrasound activationof the cluster composition was provided by a Vscan with 2 MHz probe andnominal MI of 0.8. Subsequent ultrasound exposure was applied using 500kHz custom made transducer (Imasonic SAS), 8 cycle pulses with a pulserepetition frequency of 1 kHz at MI ranging from 0.1 to 0.8.

Evans Blue

50 µl Evans Blue (50 mg/kg) was injected followed immediately by 50 µLof the cluster composition containing a nominal 1.5 µL pFMCPmicrodroplets + 4.0 µL HEPS/PFB microbubbles per mL, or 4.0 µL HEPS/PFBmicrobubbles per mL only. Activation was provided by a Vscan clinicalultrasound scanner with a 2 MHz probe for 45 seconds starting from theinjection time. This was subsequently followed by 5 minutes 500 kHzultrasound irradiation at an MI of 0.1 or 0.2. 30 minutes aftertreatment the animals were sacrificed, tissue samples; tumour, thighmuscle from the treated leg and thigh muscle from the contra lateraluntreated leg, were harvested and Evans Blue content extracted andquantified. Three animals were tested in each group, all with 45 sactivation using the VScan probe. Groups and variables are given intable 15.

TABLE 15 Groups investigated with co-injection of Evans Blue dye; USprocedure and test items. US activation was performed on all animals,three animals per group Group Subsequent US irradiation Test item 1 MI0.1, 5 min Cluster composition MI 0.2, 5 min Cluster composition 2 NoneCluster composition 3 MI 0.2, 5 min HEPS/PFB microbubbles

LiCor CW800 EPR Agent

LiCor CW800 EPR agent was administered at a dose of 5 nmol/kg bodyweight followed immediately by 50 µL of the cluster compositioncontaining a nominal 1.5 µL pFMCP microdroplets + 4.0 µL HEPS/PFBmicrobubbles per mL. Activation was provided by a Vscan clinicalultrasound scanner with a 2 MHz probe for 45 seconds starting from theinjection time. This was subsequently followed by 5 minutes 500 kHzultrasound irradiation at an MI of 0.2. Whole body epifluorescenceimaging was performed with a Pearl Impulse imaging system up to 12 hourspost administration. Animal groups and numbers are given in Table 16.

TABLE 16 Groups investigated with co-injection of LiCor CW800; USprocedures. All animals were dosed with cluster composition Group #animals Activation Subsequent US Irradiation 1 3 None None 2 4 45seconds None 3 5 45 seconds MI 0.2, 5 min

A region of interest was drawn over the tumour in the epifluorescenceimage and the mean intensity calculated. A commensurate region ofinterest was also drawn over the untreated, contralateral thigh inapproximately the same location on the leg. A dimensionless ratio wascalculated of the average image intensity in the tumour region areadivided by the average image intensity in the untreated leg. The areaunder the curve of this ratio was calculated and integrated from the 1minute to 1 hour time points.

DiR Loaded Cluster Composition

50 µL of the cluster composition containing a nominal 1.5 µL pFMCPmicrodroplets loaded with 10 mg/mL DiR dye + 4.0 µL HEPS/PFBmicrobubbles per mL was administered. Activation was provided by a Vscanclinical ultrasound scanner with a 2 MHz probe for 45 seconds startingfrom the injection time. This was subsequently followed by 5 minutes 500kHz US irradiation at an MI of 0.2. The ultrasound fields were appliedto the tumour bearing left legs of the animals. The control groupreceived the same DiR loaded cluster composition and handling proceduresbut no ultrasound exposure. Animal group details are given in Table 17.

TABLE 17 Groups investigated with injection of DiR loaded clustercomposition; animal numbers and US procedures. All animals were dosedwith cluster composition Group # animals Activation Subsequent USirradiation 1 3 None None 2 4 45 seconds MI 0.2, 5 min

Epifluorescent images were acquired with the Pearl Impulse fluorescenceimaging system both pre-injection and 1 minute post treatment(approximately 7 minutes post-injection) with standardised imageacquisitions to allow quantitative comparisons. Regions of interest weredrawn over the tumour on the left thigh, and a commensurate region ofinterest drawn on the non-tumour bearing right thigh of approximatelythe same size and anatomical location. The mean fluorescence intensityin the regions was recorded. As primary response, the difference in thefluorescence intensity between the pre-injection image and thepost-treatment image was assessed.

A two way analysis of variance was performed with factors of tumour vsnon-tumour bearing leg, and US irradiation vs no US irradiation.

E8-4 Result Evans Blue

The Evans Blue was extracted and quantified from the tissue samples(mg/mL tissue). The concentration in the treated thigh muscle wasdivided by the concentration in the untreated thigh muscle for eachanimal (matched pair) to provide a dimensionless ratio of the increaseduptake in the treated muscle. A one way ANOVA was applied to the dataand results are given in Table 18. There was a statisticallysignificant, approximate doubling in the Evans Blue uptake in the legtreated with the activated cluster composition with subsequent lowfrequency applied. For the other groups, no statistically significantincrease in uptake was observed.

TABLE 18 Uptake ratios (mean and standard deviation) for treated vs.untreated muscle tissue in different groups Group Mean SD 1 - Clustercomposition, activation and subsequent US irradiation 2.0 0.3 2 -Cluster composition and activation only 1.3 0.4 3 - HEPS/PFBmicrobubbles, activation and US irradiation 1.1 0.2

Tumour samples were taken from groups 1 and 2. The Evans Blueconcentration was divided by the concentration of Evans Blue in theuntreated thigh muscle tissue sample to provide a dimensionless ratiodescribing increase in uptake. A 2 sample t-test was applied withassumed equal sample variance. The results are shown in Table 19. Therewas an increased uptake in the tumour tissue compared to the untreatedthigh muscle of approximately 3.4 to 1 for the tumour with 500 kHzultrasound applied after activation and approximately 2 to 1 without theapplication of 500 kHz ultrasound subsequent to activation.

TABLE 19 Uptake ratios (mean, standard deviation and standard error ofmean) for treated tumour tissue vs. untreated thigh tissue in differentgroups Group Mean SD SEM 1 - Cluster composition, activation andsubsequent US irradiation 3.4 1.0 0.4 2 - Cluster composition andactivation only 2.0 0.4 0.2

Optical Imaging With LiCor CW800 EPR Contrast Agent

Typical epifluorescence images are shown in FIG. 22 for an animal fromgroup 1 (left image; activation and subsequent US irradiation), andgroup 3 (right image; no activation, no subsequent US irradiation). Thearrows indicate the location of the tumours. The images were taken withthe same Pearl imaging system scanner setting and are presented with thesame fluorescence intensity linear grey scale for direct comparison. Thetumours in the two animals are of approximately the same location andsize.

FIG. 23 shows the ratio of tumour fluorescence intensity to untreatedcontrol leg intensity from 1 minute to 9 hours post treatment. There isstatistically increased initial uptake in group 2 (squares; activationonly) compared to group 1 (diamonds; no activation, no subsequent USirradiation), and statistically increased initial uptake and uptake ratein group 3 (circles; activation and subsequent US irradiation), comparedto groups 1 and 2.

The ratio of the average intensity in the tumour region to the averageintensity in the untreated leg was calculated to create a dimensionlessTarget to Background (TBR) ratio, and the area under the TBR curve wasintegrated from 1 minute to 1 hour post treatment. Imaging was performedat 1 minute, 30 minutes and 60 minutes time points for all animals. Theresults are tabulated in Table 20. FIG. 24 shows the mean and estimatedstandard errors of groups 1, 2 and 3, labelled A, B and C respectivelyin the Figure.

TABLE 20 Area under curve (AUC) for TBR uptake ratios (mean and standarddeviation) for treated tumour tissue vs. untreated thigh tissue indifferent groups Group Mean AUC SD 1 - No activation, no subsequent USirradiation 63.7 4.5 2 - Activation only 77.0 7.3 3 - Activation andsubsequent US irradiation 94.0 7.5

An analysis of variance was applied to the three treatment groups withresulting p value of <0.001. Contrasts were applied between groups 1 and2 with p value of 0.037, and between groups 2 and 3 with p value of0.005. There is thus a statistically significant (at the 0.05 level)increase in the area under the curve between groups 1 and 2 and betweengroups 2 and 3.

DiR Loaded Microdroplet Component of the Cluster Composition

Typical post-treatment epifluorescence images are shown in FIG. 25 foran animal from group 1 receiving no ultrasound exposure (labelled A),and an animal from group 2 (labelled B) with activation and subsequentUS irradiation to the left, tumour bearing leg. The mean difference influorescence intensity defined as the post-treatment mean intensityminus the pre-injection mean intensity for the left (tumour bearing) andright legs are shown in Table 21.

TABLE 21 Mean increase ± SD in fluorescence intensity from pre-injectionvalues in different groups Group Left (tumour) leg Right leg 1 - noactivation or US irradiation -0.02 ± 0.20 0.17 ± 0.19 2 - activation andsubsequent US irradiation 2.83 ± 0.57 0.23 ± 0.31

A two way analysis of variance gives a p value <0.001 for increasedfluorescence intensity in the tumour bearing leg when ultrasoundactivation and subsequent irradiation was applied. These resultsdemonstrate localised delivery and uptake of the fluorescent dyemolecule loaded into the oil phase of the microdroplet component (C2).

E8-5 Conclusions

There was a statistically significant increase in delivery of Evans Bluedye to muscle and tumour tissue at the 0.05 level upon co-injection withthe cluster composition followed by US activation and further USirradiation. An approximate doubling of Evans Blue was observed in thetreated muscle tissue compared to no activation and no subsequent USirradiation. No significant increase in delivery of Evans Blue wasobserved with the administration of HEPS/PFB microbubbles only, andapplication of the activation and subsequent ultrasound exposure. Theuptake in tumour tissue compared to untreated muscle tissue increased bya factor of 2 upon activation only and a factor of 3.4 upon activationand subsequent US irradiation.

There was a statistically significantly increase in delivery of LicorCW800 EPR agent to tumour tissue upon co-injection with the clustercomposition and ultrasound activation compared to no ultrasoundactivation. There was a further statistically significantly increase indelivery upon subsequent US irradiation with subsequent ultrasoundirradiation for enhanced delivery, compared to activation alone.

A statistically significant increase in fluorescence intensity in thetumour bearing leg was observed when activation and subsequent USirradiation was applied after injection of a cluster composition whereDiR dye had been loaded into the oil phase of the microdroplets in C2.This demonstrates localised delivery of a molecular payload from themicrodroplets, confirming targeted spatial release and uptake to areasexposed to the ultrasound procedure.

EXAMPLE 9 (E9) - MANUFACTURE OF THE COMPONENTS AND PREPARATION OF THECLUSTER COMPOSITION

Both components were manufactured aseptically.

C1 - A raw dispersion of microbubbles were prepared from a sterile lipiddispersion and sterile gas component. The lipid dispersion was thermallysterilised in a bulk vessel and the gas was sterile filtered. Thecomplete production line was steam sterilised. The microspheres wereproduced in-situ in a colloid mill, simultaneous fed with lipiddispersion and gas. The intermediate product (raw dispersion) was thensize fractionated in a flotation vessel, diluted to target microbubbleconcentration with an aqueous solution of lyophilisation-protectingagent, filled aseptically and lyophilised.

C2 - The microdroplet emulsion was prepared from a sterile lipiddispersion and a sterile oil component. The lipid dispersion wasthermally sterilised in a bulk vessel and the oil component was sterilefiltered. The complete production line was steam sterilised. Themicrodroplets was produced in-situ in a colloid mill, simultaneously fedwith the lipid dispersion and oil component. The raw emulsion was thensize fractionated in an in-line centrifuge, diluted to targetmicrodroplet concentration with an aqueous solution of TRIS buffer, andfilled aseptically.

Three consecutive batches of each component was manufactured andsubjected to sterility testing according to Ph.Eur and USP. All sixbatches passed the sterility test.

DP - The cluster composition was prepared aseptically by reconstitutinga vial of C1 with 2 mL of C2 followed by 30 s manual homogenisation. 2mL was withdrawn from a vial of C2 using a sterile, single use syringeand needle. The content of the syringe was added through the stopper ofa vial of C1 and the resulting DP was homogenised.

What is claimed is:
 1. A method of manufacturing an embolizing agentprecursor, comprising: combining a first component contained within afirst container with a second component contained within a secondcontainer, the first component comprising a plurality of negativelycharged gaseous components and a first stabilizer, the second componentcomprising a plurality of positively charged oil components, a secondstabilizer, and a cationic surfactant; mixing the first component withthe second component such that the first and second component are heldtogether as a single agglomerated entity; adjusting the size ofindividual oil components from 1 µm to 5 µms; modifying the pH of thesecond component to 6.1-7.4; adjusting the volume concentration ofindividual oil components to a target concentration of 1-10 µlmicrodroplets/ml.
 2. The method of claim 1, further comprising removingexcess stabilizer.
 3. The method of claim 1, wherein the first componentis produced in-situ in a colloid mill, simultaneously feeding firststabilizer and gas.
 4. The method of claim 3, further comprisingremoving individual gaseous components that have a diameter less than 1µm.
 5. The method of claim 1, further comprising providing the firstcomponent in a lyophilised form.
 6. The method of claim 1, wherein thesecond component is prepared from a thermally sterilised lipiddispersion and a sterile filtered oil component.
 7. The method of claim6, wherein the second component is produced in-situ in a colloid mill,simultaneously fed with the lipid dispersion and the oil component,thereby forming a raw emulsion.
 8. The method of claim 7, wherein theraw emulsion is size fractionated in an in-line centrifuge, removingexcess stabiliser that could contaminate the attraction between firstcomponent and the second component.
 9. The method of claim 1, whereinthe agglomerated entity is stable for at least 1 hour.